Electron-Transfer Reactivity of Metalloproteins in Folded, Partially Unfolded, and Completely Unfolded Forms Scott Ichm Ael Tremain Iowa State University

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2002 Electron-transfer reactivity of metalloproteins in folded, partially unfolded, and completely unfolded forms Scott ichM ael Tremain Iowa State University

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ProQuest Information and Learning 300 North Zeeb Road, Ann Arbor, Ml 48106-1346 USA 800-521-0600

Electron-transfer reactivity of metalloproteins in folded, partially unfolded, and completely unfolded forms

Scott Michael Tremain

A dissertation submitted to the graduate faculty

in partial fulfillment of the requirements for the degree of

DOCTOR OF PHILOSOPHY

Major: Inorganic Chemistry (Biomolecular Science)

Program of Study Committee: Nenad M. Kostic, Major Professor Parag R. Chitnis Victor S.-Y. Lin John G. Verkade L. Keith Woo

Iowa State University

Ames, Iowa

2002 UMI Number: 3061872

® UMI

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This is to certify that the doctoral dissertation of

Scott Michael Tremain has met the dissertation requirements of Iowa State University

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For the j r Program iii

TABLE OF CONTENTS

CHAPTER 1. GENERAL INTRODUCTION 1

General Overview 1 Dissertation Organization 3 References 4

CHAPTER 2. FATE OF THE EXCITED TRIPLET STATE OF ZINC CYTOCHROME C IN THE PRESENCE OF IRON(III), IRON(II), IRON-FREE, AND HEME-FREE FORMS OF CYTOCHROME C 6

Abstract 6 Introduction 7 Experimental 9 Chemicals 9 Buffers 9 Proteins 9 Kinetics 10 Calculations 11 Results and Discussion 12 Natural Decay of 3Zncyt 12 Effects of Possible Quenchers on Decay of 3Zncyt 13 Possible Mechanisms of Quenching 14 Absence of Enhanced Radiationless Decay 14 Presence of Energy Transfer 15 The Question of Reductive Quenching of 3Zncyt 17 Oxidative Quenching of 3Zncyt 18 Bimolecular Rate Constants 19 Kinetic Effects of Ionic Strength 20 Conclusions 20 iv

Supplementary Material 21 Acknowledgements 21 References. 21 Tables and Figures 25 Supplementary Material 29

CHAPTER 3. MOLTEN-GLOBULE AND OTHER CONFORMATIONAL FORMS OF ZINC CYTOCHROME C. EFFECT OF PARTIAL AND COMPLETE UNFOLDING OF THE PROTEIN ON ITS ELECTRON-TRANSFER REACTIVITY 34

Abstract 34 Introduction 35 Experimental Section 39 Chemicals 39 Proteins 39 Preparation of Buffer and Dénaturant Solutions 40 Preparation of (Un)folded Forms of Zncyt 40 Circular Dichroism and Absorption Spectroscopy 41 Equilibrium Unfolding of Zncyt 41 Laser Flash Photolysis 42 Reaction Mechanism 44 Fittings of Kinetic Data 44 Results 45 Characterization of (Un)folded Forms of Zncyt by Absorption Spectroscopy 45 Equilibrium Unfolding of Zncyt 45 Characterization of (Un)folded Forms of Zncyt by Circular Dichroism Spectroscopy 45 Characterization of (Un)folded Forms of Zncyt by Natural Decay of the Triplet State 46 Conformational Transitions Between (Un)folded Forms of Zncyt 47 Demetalation of Zncyt and Remetalation of Hzcyt by Zinc(II) Ions 48 Effect of Zncyt Conformation on the Porphyrin Affinity for Zinc(II) Ions 49 V

Oxidative Quenching of 3Zncyt by Inorganic Complexes and Cyt(HI) 49 Discussion 51 Structure and Reactivity of (Un)folded Forms of Cyt(III) and Zncyt 51 Absorption and Circular Dichroism Spectra of (Un)folded Forms of Zncyt 51 Natural Decay of the Triplet State of Zncyt in Different (Un)folded Forms 53 Reactivity of 3Zncyt in the Five (Un)folded Forms 54 (A) Role of Electrostatic Interactions 54 (B) Partial Unfolding of Zncyt: The Molten-Globule Form 55

(C) Complete Unfolding of Zncyt: The Uacid and Uurea Forms 56 Conclusions 57 Supplementary Material 57 Acknowledgements 57 References 57 Tables and Figures 64 Supporting Information 75

CHAPTER 4. GENERAL CONCLUSIONS 80

APPENDIX. OPTIMIZATION OF A BACTERIAL EXPRESSION SYSTEM FOR THE SOLUBLE FORM OF TURNIP CYTOCHROME F AND ITS ISOLATION 83 Introduction 83 Experimental 86 Expression 86 Purification 88 Results and Discussion 90 Expression 90 Purification 91 Conclusions 93 References 94 Tables and Figures

ACKNOWLEDGMENTS 1

CHAPTER 1. GENERAL INTRODUCTION

General Overview

With the sequencing of the human genome complete, attention has shifted to studying

the structure and stability of proteins, protein-protein and protein-ligand interactions,

structure-function relationships, and how these relate to disease and drug discovery.1 The

biological function and reactivity of a protein depends critically on its ability to adopt a

specific and unique three-dimensional structure. When a protein is synthesized in the cell, its conformation approximates a random-coil, lacking most secondary and tertiary structure.

Utilizing only its sequence of amino acids, proteins spontaneously fold into their native conformations under physiological conditions. A protein can efficiently fold from many

unfolded states to one native state on the time scale of milliseconds to seconds.

Understanding how this process occurs is one of the great challenges in science.2"5

Metal ions and organic moieties are essential components of proteins involved in biological processes, such as respiration, metabolism, and photosynthesis. Nearly one-third of all proteins require cofactors to bind and activate substrates, transfer atoms or groups, transfer electrons, and provide structure. A class of proteins utilizing a cofactor to perform its biological function is cytochrome c. C-type cytochromes are defined as electron transfer proteins which have an iron porphyrin (heme) covalently attached to the protein backbone through cysteine residues.6 Because of their biological roles and favorable chemical and spectroscopic properties, these proteins were used in many important biochemical and biophysical studies.7"8 Especially relevant are recent studies on the folding of horse heart 2

cytochrome c.9"11

One strategy that can provide insight into the mechanism of protein folding is to

detect and characterize the intermediates in the folding pathway. One such intermediate is

the molten globule, a compact denatured form with a significant amount of nativelike

secondary structure, but largely flexible and disordered tertiary structure. In comparison with

the native folded form, the internal hydrophobic core of the molten globule is more exposed

to solvent and the side chains are more mobile.12 Characterization of these intermediates by

traditional crystallographic or NMR methods is difficult because of the dynamic,

heterogeneous, and often transient nature of these nonnative states.

Our approach involves measuring the intrinsic chemical reactivity of proteins in

various conformational forms. Cytochrome c is our protein of choice because its folding

mechanism has been thoroughly investigated and the covalently-bound heme prosthetic group provides an excellent optical marker. Various conformational forms can be captured in solution by simply changing the conditions (pH, presence of anions, presence of dénaturants).

At pH 2 in the presence of stabilizing anions, the molten-globule form of cytochrome c can be obtained.

The replacement of redox-active heme iron by redox-inactive zinc(II) in cytochrome c, allows study of reactions between heme proteins, which otherwise have overlapping absorption spectra. The long-lived triplet state of the zinc porphyrin, designated 3Zncyt, is easily produced by laser flash and is a strong reductant. Moreover, we can study photoinduced electron transfer from 3Zncyt to an electron acceptor without the complications of external reductants. 3

As a prerequisite to studying the reactivity of proteins in partially and completely unfolded forms, we investigate photoinduced electron-transfer reactions of folded 3Zncyt with various cytochrome c derivatives. There are three possible mechanisms for quenching of 3Zncyt: enhancement of radiationless decay, Fôrster (dipole-dipole) energy transfer, and electron transfer. Our work will provide insight into the mechanism for quenching of 3Zncyt by the iron(IH), iron(II), iron-free, and heme-free forms of cytochrome c under conditions in which these proteins are folded.

To study the effect of protein conformation on reactivity, we compare the electron-

3 transfer properties of Zncyt in the folded forms at low (Fiow) and high (Fhigh) ionic strength, molten-globule (MG), and forms unfolded by acid (Uacid) and by urea (Uurea) toward the

3 3+ following four oxidative quenchers: Fe(CN)6 ~, Co(acac)3, Co(phen>3 , and iron(III) cytochrome c. The observed bimolecular rate constants will provide insights into the effect of the protein matrix in modulating electron-transfer reactivity.

Dissertation Organization

Chapter 2 explores the fate of the excited triplet state of zinc cytochrome c in the presence of iron(IH), iron(II), iron-free, and heme-free forms of cytochrome c. The mechanism for quenching of the excited triplet state of zinc cytochrome c in the completely folded form is determined. Chapter 3 is a comprehensive study of the effect of protein conformation on electron-transfer reactivity. An electroneutral transition metal complex assesses the consequences of porphyrin exposure on electron-transfer reactivity. Chapters 2 and 3 represent papers that have been published in peer-reviewed journals. The dissertation 4

ends with Chapter 4, which gives the overall conclusions. The Appendix provides details on

the expression and isolation of the soluble form of turnip cytochrome / for use in future studies.

References

(1) Wittung-Stafshede, P. Acc. Chem. Res. 2002, 35, 201-208.

(2) Rumbly, J.; Hoang, L.; Mayne, L.; Englander, S. W. Proc. Natl. Acad. Sci. U.S.A. 2001,

98, 105-112.

(3) Dobson, C. M.; Karplus, M. Curr. Opin. Struct. Biol. 1999, 9, 92-101.

(4) Ellis, J. R.; Hartl, F. U. Curr. Opin. Struct. Biol. 1999, 9, 102-110.

(5) Onuchic, J. N.; Luthey-Schulten, Z.; Wolynes, P. G. Annu. Rev. Phys. Chem. 1997,48,

545-600.

(6) Cytochrome c: A Multidisciplinary Approach, Scott, R. A.; Mauk, A. G., Eds.;

University Science Books: Sausalito, CA, 1996.

(7) Pettigrew, G. W.; Moore, G. R. Cytochromes c. Biological Aspects; Springer-Verlag:

Berlin, 1987.

(8) Moore, G. R.; Petti grew, G. W. Cytochromes c. Evolutionary, Structural and

Physicochemical Aspects', Springer-Verlag: Berlin, 1990.

(9) Ramachandra Shastry, M. C.; Sauder, J. M.; Roder, H. Acc. Chem. Res. 1998, 31, 717-

725.

(10) Yeh, S.-R.; Han, S.; Rousseau, D. L. Acc. Chem. Res. 1998, 31, 727-736. 5

(11) Telford, J. R.; Wittung-Stafshede, P.; Gray, H. B.; Winkler, J. R. Acc. Chem. Res. 1998,

31,755-763.

(12) Goto, Y.; Takahashi, N.; Fink, A. L. Biochemistry 1990, 29, 3480-3488. 6

CHAPTER 2. FATE OF THE EXCITED TRIPLET STATE OF ZINC CYTOCHROME C IN THE PRESENCE OF IRON(III), IRON(II), IRON-FREE, AND HEME FREE FORMS OF CYTOCHROME C

A paper published by and reprinted with permission from

Inorganica Chimica Acta 2000, 300-302, 733-740

Scott M. Tremain and Nenad M. Kostic

Abstract

We study, by laser flash photolysis, the mechanism of quenching the triplet state of zinc cytochrome c, 3Zncyt, by the iron(IH), iron(H), iron-free, and heme-free forms of cytochrome c. The method of inserting zinc(II) ion into the heme group in a protein overcomes the difficulties in studying redox reactions between two heme proteins having similar absorption spectra. Quenching of positively charged 3Zncyt by these positively charged reactants is promoted at high ionic strength, n = 1500 mM, which shields electrostatic charge and weakens protein-protein repulsion. Upon addition of potential quenchers, the rate constant for 3Zncyt decay becomes higher in the following order: ferrocytochrome c < iron-free cytochrome c « ferricytochrome c. However, upon addition of as much as 350 pM heme-free cytochrome c, the rate of 3Zncyt decay remains unchanged.

The bimolecular rate constants for the reactions of 3Zncyt are as follows: (1.5 ± 0.2) x 104 7

M-1 s-1 with ferrocytochrome c, (3.3 ± 0.2) x 104 M-1 s-1 with iron-free cytochrome c, and

(5.1 ±0.3) x 106 M-1 s-1 with ferricytochrome c. Oxidative quenching of 3Zncyt by

ferricytochrome c is proven by the observed formation of zinc cytochrome c cation radical.

Energy transfer is responsible for the weak quenching of 3Zncyt by iron-free cytochrome c

and ferrocytochrome c, as evident in the lack of any observable transient radical-ions Zncyt+

and Zncyt" and the good overlap of the emission band of 3Zncyt with the absorption bands of

ferrocytochrome c and iron-free cytochrome c.

Introduction

Because metalloproteins act as electron carriers and redox enzymes in many

biological processes, chemical mechanisms of their electron-transfer reactions are being studied vigorously [1-5]. Various cytochromes c are ubiquitous in biological systems as prototypical electron carriers. Because of their biological roles and favorable chemical and spectroscopic properties, mammalian cytochromes c were used in numerous biochemical and biophysical studies of general importance [6-8]. Their oxidoreduction reactions with various chemical and biological agents have been investigated. Three-dimensional structures of ironCHI) and iron(H) forms of cytochromes c in both crystal and solution are known in detail

[7,9-11]. The highly positively charged protein reacts with external redox partners via an exposed heme edge, which is surrounded by positively charged lysine residues [7].

Motivation for this work comes from the problem of studying the thermal redox reaction between two heme proteins. Because their absorption spectra overlap, changes in their oxidation states cannot be easily monitored spectrophotometrically [12,13]. A solution 8

to this problem is to replace redox-active iron in the heme by redox-inactive zinc(II), so that

the redox step becomes photoinduced. Replacement of iron(II) by zinc(H) does not

significantly perturb the structure of cytochrome c [14-16] and its interactions with other

proteins [17-19], Derivatives of cytochrome c containing various metals in the place of iron

have been prepared and characterized [20]. This method has proven useful in the study of

some other physiological partners, notably cytochrome c and its peroxidase [2]. Cytochrome

c noninvasive^ reconstituted with zinc(II), called simply zinc cytochrome c and designated

Zncyt [14-17,21], has been used in kinetic studies of electron-transfer reactions in our and

other laboratories [16,22-40]. It has absorption maxima at 423 (Soret), 549, and 585 nm; a

fluorescent (singlet) excited state with the lifetime of 3.2 ns; and fluorescence maxima at 590

and 640 nm. Especially useful for kinetic studies is the lowest-lying triplet excited state, designated 3Zncyt. Its lifetime is between 7 and 15 ms, depending on the protein purity.

Because this lifetime is very long, electron-transfer reactions of the triplet state can be studied relatively easily. The 3Zncyt state is a much stronger reductant than the native protein.

Because this state can be created simply by a laser pulse, no external reducing agents are needed, and kinetic treatments can be accurate.

In this work we explore the effects of different possible quenchers on the lifetime of

3Zncyt. Photoinduced reactions of 3Zncyt with iron(HI) cytochrome c (Felucyt), iron(II) cytochrome c (Fe"cyt), iron-free cytochrome c (Hzcyt), and heme-free cytochrome c (apocyt), at increasing ionic strength provide insight into the mechanism of possible quenching for each of these reactants. Although these are not biological reactions in the narrow sense, this work will be relevant to the future study of reactions between heme proteins. 9

Experimental

Chemicals. Distilled water was demineralized to a resistivity greater than 17 Mfi*cm by the Bamsted Nanopure n apparatus. Chromatography resins and gels were purchased from Sigma Chemical Co., Pharmacia, and Bio-Rad. Hydrogen fluoride, nitrogen, and ultrapure argon were purchased from Air Products Co. All other chemicals were purchased from Fisher Chemical Co. and used as received.

Buffers. All the buffers were prepared fresh from the solid salts NaH2P04*H20 and

Na2HP04*7Hz0 and had pH values of 7.00±0.05. The ionic strengths (p) higher than 10 mM were achieved by addition of solid NaCl. All buffers are characterized with ionic strength, not concentration. Buffers were degassed thoroughly prior to use by bubbling with wet ultrapure argon.

Proteins. Horse-heart cytochrome c was obtained from Sigma Chemical Co.

Iron(in) and iron(H) forms were prepared simultaneously by treatment with enough ascorbic acid to reduce approximately one half of the protein in the sample. Separation was carried out with a CM-52 ion-exchange column; upon increasing the ionic strength, iron(H) form eluted first. The iron(m) and iron(H) forms were also prepared separately, by treatment with an excess of K3[Fe(CN)6] or ascorbic acid, and desalted with a Bio-Rad Econo-Pac 10 DG column. The rates of quenching 3Zncyt by Fen,cyt and Fe"cyt were independent of the method of preparation. The concentration of purified Fe"cyt or Fen,cyt was determined spectrophotometrically; the molar absorptivity difference between reduced and oxidized cytochrome c at 550 nm is 18.5 mM-1 cm"'.

Zinc cytochrome c was prepared and purified by a modification [16] of the original 10 procedure [17,40], A saturated solution of NazHPO, was used instead of solid Na]HP04 to adjust pH to 6.0 after reconstitution with zinc(II) ions. This method improves the yield of

Zncyt by minimizing the loss of protein embedded within the precipitate. All procedures were done in the dark and as quickly as possible. Two of the criteria for purity were the absorbance ratios A423/A549 > 15.4 and A549/A585 < 2.0. Another criterion for purity was the natural decay of 3Zncyt, < 110 s-1. Iron-free (so called free-base) [14] and heme-free

[41,42] forms of cytochrome c were prepared and purified by standard procedures.

Concentration of apocyt was determined spectrophotometrically at 595 nm, with a Bio-Rad protein assay.

Kinetics. Laser-flash photolysis on the ps time scale was performed with a standard apparatus [27]. A Phase-R (now Luminex) DL 1100 laser contained a 50 gM solution of the dye rhodamine 590 in methanol and delivered 0.4-gs pulses of excitation light. The sample solution in a 10-mm cuvette was thoroughly deaerated by gentle flushing with wet ultrapure argon through a needle held 5 mm above the solution for at least 15 minutes after each addition of quencher or until the rate constant for 3Zncyt decay ceased changing. This procedure prevents denaturation of protein that might occur upon bubbling gas through the solution and ensures complete deoxygenation. Owing to some evaporation of the solvent in the argon stream, the sample was weighed before and after degassing to obtain accurate concentrations of Zncyt and quencher.

Decay of 3Zncyt was monitored at 460 nm, where its transient absorbance reaches a maximum. Eight to ten pulses were recorded for each addition of quencher. Appearance and disappearance of the zinc cytochrome c cation radical, Zncyt+, was monitored at 675 nm, 11

where the difference in absorbance between Zncyt* and 3Zncyt is greatest. Temperature was

held by a circulating bath at 25.0 ± 0.1°C. The change of absorbance with time was analyzed

with SigmaPlot v. 4.0. The results from separate fittings of the traces obtained by successive

flashes were averaged by the least-squares method.

The concentration of Zncyt was always 10 pM. The concentration of 3Zncyt

depended on the excitation power and was ca. 1.0 pM. The mole ratio of quencher to 3Zncyt

was greater than 10:1, so that the conditions for pseudo-first-order reaction always prevailed.

Second-order rate constants were obtained from the corresponding pseudo-first-order rate

constants by least-squares fittings.

Calculations. The rate constant kea for energy transfer by the Fôrster (dipole-dipole)

mechanism was calculated [43] according to Eq. (1), where fc is dipole-dipole orientation

factor, n is refractive index of the medium, ris natural lifetime of the donor, and R is center-

to-center distance between donor and acceptor (in cm). The overlap integral (in cm6 mol-1)

(8 8 )lr K, - - ^ jF„a)eA(A)AV/1 (1)

describes the interaction between donor emission and acceptor absorption bands; Fd(A) is the

normalized emission probability of the donor; and £a(A) is the molar decadic extinction spectrum of the acceptor (cm2 mol-1). Numerical data for the donor (3Zncyt) emission were obtained by scanning the published spectrum [17] into a computer, manually obtaining the xy coordinates for 90 points (ca. one point per nm) with Adobe PhotoShop 4.0, and converting those xy coordinates into wavelengths and emission intensity. See Fig. SI in the supplementary material. Numerical data for the acceptor (Fenlcyt, Fe"cyt, and Hzcyt) 12 absorption were obtained by converting their measured spectra into ASCII files and importing the data into SigmaPIot v. 4.0. The numerical value of the overlap integral, shown

m in Fig. S2 in the supplementary material, is calculated for H2cyt, Fe"cyt, and Fe cyt [44].

The effect of ionic strength is analyzed by Br0nsted-Debye-Huckel theory, often used to estimate charges of small, globular proteins, as in Eq. (2), where symbols k and ko are

1 + kR1 1+ KR2 1+ K/^ bimolecular rate constants at ionic strengths |i and zero; Z, and Z? are net charges of the reactants, and and Z?2 are their radii; Rt is the radius of the transition state for the bimolecular reaction; a = 1.17 in water at 25°C; and K = 0.329 \iA Â-'. Under assumption R\

= Rz = Ravi Eq. (2) reduces to the widely used Eq. (3), which is used also in this study. The

In* = ln*„ + 2Z'Z^'/" (3)

radius of cytochrome c is 18.5 Â [45], so /?av = 18.5 Â.

Results and Discussion

Natural Decay of 3Zncyt. The excitation by laser of the porphyrin ^-system can be considered a promotion of an electron with inversion of its spin. Because the excited (triplet) and ground (singlet) states differ in spin multiplicity, the lifetime of 3Zncyt is long. The rate constant kd for this simple decay in Eq. (4) was obtained from fittings of the traces to the

3 Zncyt ——> Zncyt (4) monoexponential function in Eq. (5). In the absence of added quencher, = 80 ± 5 s-1, 13

AA = ae~kit (5) independent of Zncyt concentration, ionic strength in the interval 2.5 mM < g. < 1500 mM, and wavelength. This rate constant remained unchanged upon separate additions of 1.0 mM ascorbic acid and 1.2 mM sodium dithionite, as reductants.

Effects of Possible Quenchers on Decay of 3Zncyt. To ensure complete removal of

3 3 [Fe(CN)6] ~ ions, the unwanted quencher of Zncyt, a control experiment was carried out with horse-heart cytochrome c containing 90% Femcyt and 10% Fe"cyt, as determined spectrophotometrically. Identical bimolecular rate constants for quenching of 3Zncyt were obtained in experiments with the commercial and desalted preparations of FelI,cyt. Although the [Fe(CN)ô]3~ ion is used as an oxidant for cytochrome c, it is completely removed by purification or desalting and will not interfere with the quenching of 3Zncyt in subsequent experiments.

At low ionic strength, g = 10 mM, in the presence of concentrations as high as 140

3 gM Fe'"cyt, 120 gM Fe"cyt, 200 piM H2cyt, and 200 |iM apocyt, the rate of Zncyt decay remained unchanged. At high ionic strength, |X = 1500 mM, the 3Zncyt decay became faster and remained exponential upon incremental addition of Fe'"cyt up to 140 gM, Fe"cyt up to

150 nM, and H2cyt up to 240 gM. However, upon addition of apocyt even up to 350 gM, the rate of 3Zncyt decay remained unchanged. Typical traces are shown in Figs. S3 and S4 in the supplementary material. The pseudo-first-order rate constant was directly proportional to the quencher concentration and depended on ionic strength. In no case did we detect saturation behavior (leveling off), even at the highest concentrations of quencher mentioned above. The bimolecular rate constants kbim, given in Table 1, are obtained from the slopes of the linear 14

plots in Fig. 1.

Possible Mechanisms of Quenching. There are three possible mechanisms for

quenching of 3Zncyt: enhancement of radiationless decay, Fôrster (dipole-dipole) energy

transfer, and electron transfer. Enhanced radiationless decay is the slight increase in rate of

3Zncyt decay attributed, without evidence, to a slight conformational change in Zncyt upon

association with another protein [30,46], Energy transfer can occur if the emission bands of

3Zncyt (donor) at 590, 640, and 736 nm overlap with the absorption bands of the potential

acceptor. Electron transfer can result in oxidative (Eq. (6)) or reductive (Eq. (7)) quenching

3 Zncyt+Q > Zncyt+ +Q (6)

3 Zncyt + Q » Zncyt" + Q+ (7)

by various quenchers, designated Q. The products of these reactions are the ion radicals

Zncyt* and Zncyt-. To our knowledge, the reaction in Eq. (7) has been studied only in this

laboratory [20].

Absence of Enhanced Radiationless Decay. In order to determine if enhanced radiationless decay plays a role in the quenching of 3Zncyt, apocyt was used as a possible quencher. Lacking heme, this protein does not absorb wavelengths greater than 280 nm and is incapable of electron transfer and energy transfer. At low ionic strength, g = 10 mM, the rate of 3Zncyt decay does not increase as the concentration of apocyt is raised to 200 fiM.

The bimolecular rate constant remains < 1 x 104 M-1 s-1, below the threshold of our kinetic method. At high ionic strength, g = 1500 mM, the rate of 3Zncyt decay still does not increase even upon addition of 350 gM apocyt (see Fig. 1), and the bimolecular rate constant remains 15

< 1 x 104 M-1 s-1. These control experiments with apocyt show that enhanced radiationless

decay is not an efficient mechanism of quenching 3Zncyt. Even at high ionic strength, when

electrostatic repulsions between positively charged Zncyt and apocyt are minimized, these

proteins do not associate to an observable extent. Enhanced radiationless decay will not be

n m an efficient mechanism of quenching in the cases of H2cyt, Fe cyt, and Fe cyt, either. In the

cases where enhanced radiationless decay of 3Zncyt was observed, in our and other

laboratories, this mechanism was proven by the same criteria applied in this study [26,29,46].

Presence of Energy Transfer. For energy transfer to be an efficient mechanism of

quenching 3Zncyt, the emission spectrum of the 3Zncyt (donor) and the absorbance spectrum

of the acceptor must overlap. Both Fe"cyt and H2cyt have absorption bands that may overlap

the emission bands of 3Zncyt at 590,640, and 736 nm. The sharp absorption band of Fe"cyt

at 550 nm overlaps slightly with the emission band of 3Zncyt at 590 nm. Broad absorption

3 bands of H2cyt at 568 and 620 nm overlap well with the emission bands of Zncyt at 590 and

640 nm. Therefore the rate of energy transfer is expected to be larger for H2cyt than for

Fe"cyt. At low ionic strength, jo. = 20 mM for Fe"cyt and g = 10 mM for H2cyt, the rate of

3Zncyt decay is not affected by the increasing concentration of Fe"cyt up to 120 |xM and of

Hicyt up to 200 |iM. However, at high ionic strength, g = 1500 mM, the rate of 3Zncyt decay increases as the concentration of Fe"cyt or H2cyt is raised to 150 |iM and 240 (iM, respectively; see Fig. 1. The bimolecular rate constant for H2cyt is twice as large as that for

Fencyt; although small, this difference is fully reproducible. At high ionic strength, electrostatic repulsions between these proteins are minimized, allowing the observed quenching of 3Zncyt to occur. On the basis of these results and the control experiments with 16

apocyt, we conclude that energy transfer, not enhanced radiationless decay or electron

transfer, is the most efficient mechanism of quenching 3Zncyt by Feucyt and Hzcyt.

To further verify energy transfer as the mechanism of quenching 3Zncyt, we

m calculated, using Eq. (1), the rate constant for Fe cyt, Fe"cyt, and H2cyt. We set R = 25

Â, n = 1.4, r= 75 ms [47], and K= 1, and assumed 100% overlap between donor emission,

which occurs from 570 to 670 nm [17] and acceptor absorption at room temperature (r.t.).

-1 m The results (ken values) were 205, 38, and 385 s for Fe cyt, Fe"cyt, and Hjcyt, respectively.

The ability of Fe"cyt and Hicyt to quench 3Zncyt by energy transfer is confirmed. Moreover,

energy transfer also contributes slightly to the quenching of 3Zncyt by Feulcyt.

A general mechanism for the reaction between electron donor D and the electron

acceptor A is shown in Eq. (8). The quotient W^off is the association constant K\. Under

D + A ===== DA k » D+A" (8) off

the so-called improved steady-state approximation and the condition [A]»[D], Eq. (9) is

k — /n\ 01,5 *„n [A] + ktff + kf

obtained [48]. In the limiting case when koff >Ar on[A] + kf, Eq. (9) yields Eq. (10). In this

*cs=*AMA] = *bim[A] (10) case, common in studies of protein reactions, the observed rate constant linearly depends on the concentration of the reactant present in excess. The symbol fcbim represents the bimolecular rate constant for the reactions of 3Zncyt with various quenchers. 17

Calculated values for koa in the reactions of 3Zncyt with H%cyt and with Fencyt are 5.1

x 107 s~l at n = 2.5 mM and 8.2 x 10* s-1 at n = 1500 mM. We fitted to Eq. (9) experimental

3 values of *obs for quenching of Zncyt by H2cyt and Fe"cyt. Justifiably, koa and Ar0ff were

6 -1 fixed. For these reactants, which bear large positive charge, the parameters k0a > 2 x 10 s

3 -1 and KA< 4 x 10 M are quite reasonable. The km values obtained by two methods,

embodied in Eqs. (1) and (9), agreed well.

The Question of Reductive Quenching of 3Zncyt. The bimolecular rate constant

for the reductive quenching reaction in Eq. (11) at pH 6.5 and ionic strength of 5.0 mM is

3Zncyt + EDTA > Zncyt" + EDTA+ (11)

1 x 104 M-1 s '. Rapid decomposition of the cation radical EDTA+ suppresses the reaction in

Eq. (12), and the transient anion radical Zncyt" was detected at 690 nm [20]. It was

Zncyf + EDTA+ » Zncyt + EDTA (12) previously shown [20] that Fe"cyt does not quench 3Zncyt at pH 7.0 and ionic strength of 40 mM, probably because both reactants bear the net charge of +6 under these conditions.

Furthermore, the electron self-exchange reaction of the native (iron) protein is relatively slow

[7]. These studies were the background to this one.

We re-examined the reaction in Eq. (11) and found that the transient Zncyt" is detectable between 650 and 700 nm and best evident at 680 nm. Knowing this, we checked if 3Zncyt is affected by the possible reductive quencher Fencyt present in large excess. At low ionic strength, |x = 20 mM, the rate of 3Zncyt decay remains unchanged, and the transient

Zncyt is not observed. At high ionic strength, p = 1500 mM, the rate of 3Zncyt decay 18 increases upon raising the concentration of Feucyt to 150 (jM. There is, however, no evidence of the transient Zncyt" in the range 650-710 nm. These results argue against, but do not conclusively refute, reductive quenching of 3Zncyt by Fe"cyt. The more likely mechanism of quenching 3Zncyt remains energy transfer.

Oxidative Quenching of 3Zncyt. We studied the reaction in Eq. (13) at pH 7.0 and

3 Zncyt + Fe'ncyt > Zncyt* + Fe"cyt (13) at increasing ionic strengths. In the presence of Femcyt the decay of 3Zncyt became faster as ionic strength was raised from 2.5 to 1500 mM but remained monoexponential. The rate constants for disappearance of 3Zncyt and for appearance of Zncyt1" were equal, within the error margins. The formation of Zncyt"1" is direct evidence for oxidative quenching, as in Eq.

(13). The transient Zncyt"1" signal was evident between 660 and 690 nm, and 670 nm was the best wavelength to record it. The pseudo-first-order rate constant for the reaction in Eq. (13) is directly proportional to Femcyt concentration and dependent on ionic strength, as shown in

Fig. S5 in the supplementary material. No saturation is observed at high Femcyt concentration, and the plots go through the origin. At low ionic strength, p. = 2.5 mM, there is no increase in the rate of 3Zncyt decay as the concentration of Fe'"cyt is raised to 150 jiM.

Evidently, the bimolecular rate constant is less than 1 x 104 M"1 s"'. There is no evidence for the formation of the transient Zncyt* at 670 nm, as shown in Fig. 2(a). These results show that at low ionic strength, protein-protein repulsion dominates and electron transfer does not occur. At high ionic strength, n = 1500 mM, a large increase is observed in the rate of 3Zncyt decay as the concentration of Feracyt is raised to 140 |iM. The bimolecular rate constant is 19

5.1 x 106 M-1 s-1. Fig. 2(b) shows formation of the transient Zncyt+, clear evidence for oxidative quenching. Although energy transfer is a viable mechanism of quenching, calculations show that it contributes only ca. 5% to the quenching of 3Zncyt by Femcyt.

Bimolecular Rate Constants. Table 1 shows that as ionic strength increases, so does the rate of 3Zncyt decay. This trend is most evident in the reaction with Fe"'cyt. Shielding of the charged groups by counter ions allows for protein collision. The mechanism of quenching is purely diffusional, with no preformed protein-protein complexes present even at high concentration of the quencher. The near absence of quenching of 3Zncyt by Feucyt,

Hzcyt, and apocyt shows that oxidative quenching is more efficient than energy transfer, enhanced radiationless decay, and reductive quenching under these conditions.

We confirm a previous report that the reaction of 3Zncyt with Fe"cyt in the presence of 10 mM phosphate buffer at pH 7, has an apparent bimolecular rate constant of 1 x 104 M~l s"1 [49]. This previous study also found that the reaction of 3Zncyt with Femcyt in the presence of 10 mM phosphate buffer at pH 7, had an apparent bimolecular rate constant of

2.4 x 106 M-1 s-1. Our study, however, found no evidence of quenching at this low ionic

4 _I strength, ^ = 20 mM; our results indicate kbim < 1 x 10 M~'s . The ionic strength must be close to 500 mM for the charges to be sufficiently shielded and the bimolecular rate constant to reach 2.0 x 106 M-1 s~l. One possible explanation of the discrepancy is that [Fe(CN)ô]3- ion, used as an oxidant in preparing Femcyt, was incompletely removed before the kinetic experiments in the previous study. In our control experiments, [Fe(CN)e]3- concentration as low as 5 x 10~8 M could account for the large apparent bimolecular rate constant reported 20

before. This concentration is approximately 1% of that used for oxidizing cytochrome c prior

to the kinetic experiments. In future studies of the interaction of 3Zncyt with various electron

acceptors, one should be aware of contaminants such as Feucyt and Hicyt, two redox-inactive

proteins that could be present in preparations of Zncyt, since these proteins quench 3Zncyt by

energy transfer.

Kinetic Effects of Ionic Strength. The effect of ionic strength on the reaction of

3Zncyt and Fenicyt (Table 1 and Fig. S5) is analyzed by Brgnsted-Debye-Huckel theory, as in

Eqs.(2) and (3). The plot in Fig. 3 has a slope of 60 ± 2. The fitted overall charges, Z, = 5.6

± 0.2 for Feulcyt and Z, = 4.6 ± 0.2 for Zncyt, deviate from the known charges of +7 and +6,

respectively. These results are typical for reaction of two positively charged molecules in

which monopole-monopole interactions dominate.

Conclusions

Substitution of iron ion by the redox-inactive zinc(H) ion allows study of reactions

between heme proteins, which have overlapping absorption spectra. Experiments with

lu 3 Fe cyt, Fe"cyt, H2cyt, and apocyt revealed the mechanism of quenching Zncyt. High ionic strength is necessary for avoidance of protein-protein repulsion. Electron transfer from

3Zncyt to Fen'cyt is a more efficient quenching mechanism than energy transfer and enhanced

n 3 radiationless decay at high ionic strength. Iron-free H2cyt and also Fe cyt quenches Zncyt by energy transfer, Hicyt is more efficient owing to a better overlap of its absorption bands with the emission bands of 3Zncyt. This work will be relevant to the future study of reactions between heme proteins. 21

Supplementary Material. Five Figures, showing kinetic traces of 3Zncyt in the

u 3 absence and presence of Fe"'cyt, Fe cyt, H2cyt, and apocyt; emission spectrum of Zncyt at

r.t.; plots illustrating the determination of the overlap integral necessary for calculation of

energy-transfer rate constant *e„ by Eq. (1); and the dependence of kobs on ionic strength.

Acknowledgements. This study was supported by the US National Science

Foundation through Grant MCB-9808392.

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Tables and Figures

4 -1 -1 3 Table 1. Bimolecular Rate Constants (fcbim x 10" , M s ) for the Reaction Zncyt with

Possible Quenchers at pH 7.0 and 25°Ca

Quencher 2.5 10 20 40 475 500 1500

Feulcyt c 0.7±0.8 1±3 1±1 23±6 205±9 286±14 513+30

Fencyt c 0.7±0.7 19+3 15±2

H2cytc [-1+4] 33±3

Apocytc [-1.210.9] 0.15±0.08

Ascorbic [-1.010.1] acid Sodium [-0.2+0.7] dithionite a Small negative values arise when slopes of pseudo-first-order plots deviate slightly from the horizontal 26

a Fe"'cyt

• H2cyt • Fe"cyt • apocyt

Concentration of potential quencher / jiM

Fig. 1. Dependence on protein concentration of the rate constant k0bS for the reaction of 3 ul n Zncyt with Fe cyt, H2cyt, Fe cyt, and apocyt at an ionic strength of 1.500 M, pH 7.0, and 3 25°C. Rate of natural decay of Zncyt kd is subtracted from Z:obs. The solid lines are fittings to Eq. (10); their slopes yield kbim. 27

0.015 -

0.010 -

0.005 -

0.015 -

0.010 -

0.005 -

0.000 -

0.00 0.01 0.02 0.03 0.04 0.05 0.06 Time / s

Fig. 2. Decay of 3Zncyt in the presence of 80 |aM Fe'"cyt at ionic strengths of (a) 2.5 mM and (b) 1.500 M. The solid lines are fittings to (a) monoexponential and (b) biexponential equations. In (a), k\ = 80 s"1, showing that 3Zncyt is not quenched by Fenicyt at low ionic strength. In (b), k\ = 380 s"' and &2 = 50 s~\ corresponding to fast growth and slow decay of transient Zncyt* formed upon reaction of 3Zncyt with Feulcyt at high ionic strength. 28

20 -1

15 -

S 10 - 4C ç

5 -

0 - 0.00 0.05 0.10 0.15

1 +6.09/j1/2

Fig. 3. Dependence on ionic strength of the bimolecular rate constants &bim for the reaction of 3Zncyt with Femcyt. The solid line is the fitting to Eq. (3). 29

Supplementary Material

0.0010

5" 0.0008

a 0.0006

c 0.0004 œ E 0.0002 /

0.0000 540 560 580 600 620 640 660 680 Wavelength (nm)

Fig. SI. Emission spectrum of 3Zncyt obtained manually by plotting light intensity versus wavelength (ca. 1 point per nm) using scanned picture of a published room temperature emission spectrum [17]. 30

26-14

16-14 •

36-14

18000 17500 17000 16500 16000 15500

Wavelength (cm"')

Figure S2. Plots of Fo versus wavelength for determination of overlap integral, which is necessary for calculation, using Eq. (1), of long-range energy-transfer rate constant ktn. Plots show overlap of 3Zncyt emission spectrum with (a) H%cyt, (b) Fency, and (c) Fen,cyt absorption spectra. Area under the curves, determined by integration, provides the overlap integral. 31

—I 1 1 1 1 1 1 1— 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 Time (ms)

Fig. S3. Kinetic traces showing decay of 3Zncyt monitored at 460 nm in the (a) absence and (b) presence of 80 pM Femcyt. Experimental conditions are |i = 1500 mM, pH 7, and 25°C. 32

1.0 -

0.8 -

** 0.6 -

0.4 - 1 K,\ 0.2 - (a) (b) (c) (d) 0.0 -

10 15 20 25 Time (ms)

Fig. S4, Kinetic traces showing decay of 3Zncyt monitored at 460 nm in the absence and presence of various potential quenchers: (a) apocyt, (b) absence of quencher, (c) Fencyt, and (d) Hzcyt. Experimental conditions are protein concentration = 150 pM, p. = 1500 mM, pH 7.0, and 25°C. Smooth, solid line is monoexponential fitting of the decay of 3Zncyt in presence of 150 fiM Fe'"cyt to illustrate the most efficient mechanism of quenching, oxidative electron transfer. 33

1000

1500 mM >> « 900 Linear Regression o 500 mM 4) Q Linear Regression 800 474 5 mM Linear Regression 40.9 mM *£ 700 Linear Regression 2.5 mM Linear Regression

200 -

[Fe'"cytcl (pM)

3 n Fig. S5. Dependence on Fe'"cyt concentration of kobs for the reaction of Zncyt with Fe' cyt 3 at increasing ionic strength. Rate of natural decay of Zncyt kd is subtracted from Ârobs. The solid lines are fittings to Eq. (10), whose slopes yield jfcbim. 34

CHAPTER 3. MOLTEN-GLOBULE AND OTHER

CONFORMATIONAL FORMS OF ZINC CYTOCHROME C.

EFFECT OF PARTIAL AND COMPLETE UNFOLDING OF

THE PROTEIN ON ITS ELECTRON TRANSFER

REACTIVITY

A paper published by and reproduced with permission from

Inorganic Chemistry 2002, 41, 3291-3301

Scott M. Tremain and Nenad M. Kostic

Abstract

To test the effect of protein conformation on reactivity, we use laser flash photolysis to compare the electron-transfer properties of the triplet state of zinc-substituted cytochrome

3 c, designated Zncyt, in the folded forms at low (Flow) and high (Fhigh) ionic strength, molten- globule (MG) form, and the forms unfolded by acid (Uacid) and urea (Uurea) toward the following four oxidative quenchers: FefCN)^3"", Co(acac)s, Co(phen)33+, and iron(HI) cytochrome c. We characterize the conformational forms of Zncyt on the basis of the far-UV circular dichroism, Soret absorption, and rate constant for natural decay of the triplet state.

This rate constant in the absence of quencher increases in the order Fhigh < Flow < MG < UaCid

< Uurea because the exposure of porphyrin to solvent increases as Zncyt unfolds. Bimolecular 35

rate constants for the reaction of 3Zncyt with the four quenchers show significant effects on

reactivity of electrostatic interactions and porphyrin exposure to solvent. This rate constant at

the ionic strength of 20 mM increases upon unfolding by urea and acid, respectively, as

follows: 1340-fold and 466-fold when the quencher is Co(phen)33+ and 168-fold and 36-fold

3 when the quencher is cyt(III). To compare reactivity of Zncyt in the F|0W, F^, MG, UaCjd,

and Uurea forms without complicating effects of electrostatic interactions, we used the

electroneutral quencher Co(acac)3. Indeed, reactivity of folded 3Zncyt with Co(acac)3 was

independent of ionic strength. Reactivity of 3Zncyt with Co(acac)3 upon partial and complete

unfolding increases 10-fold, 54-fold, and 64-fold in the molten-globule, urea-unfolded, and

acid-unfolded forms.

Introduction

Because metalloproteins act as electron carriers and redox enzymes in many

biological processes, chemical mechanisms of their electron-transfer reactions are being

studied vigorously.1-6 Various cytochromes c are ubiquitous in biological systems. Because

of their biological roles and favorable chemical and spectroscopic properties, these proteins

were used in many important biochemical and biophysical studies.7-9

Conformational flexibility of proteins is essential for their function.10,11 Despite

much current research, however, little is known about the thermodynamics and kinetics of

protein denaturation and unfolding.12-14 Various experimental studies gave evidence for the conformational fluctuations and heterogeneity of proteins.15-20 Partially unfolded forms are

presumed intermediates in the folding of denatured proteins and seem to be implicated in 36 diseases caused by protein misfolding.21-24 We are interested in the effects of these conformational changes on the chemical reactivity of proteins.

Horse heart cytochrome c, whose oxidation states are designated cyt(H) and cyt(IH), is often used because of its well-defined structure8,25-28 and suitable spectroscopic properties.

The heme group, covalently attached to the backbone through cysteine residues 14 and 17, interacts with the surrounding protein. The six-coordinate low-spin iron binds His18 and

Met80 as axial ligands. Polar interactions between the propionyl substituents in the heme and nearby polar side chains and hydrophobic interactions between the porphyrin ring and buried nonpolar side chains abound in the native structures. Electron transfer with external redox partners occurs via an edge of the heme that is partially exposed at the protein surface and surrounded by positively charged lysine residues.8

At least five distinct forms of iron cyochrome c, dependent on pH, have been observed spectroscopically.29-31 At neutral pH, the folded low-spin form with Met80 and

HislS as ligands dominates. At pH above 8-10, the dominant species is the low-spin so- called alkaline conformation, in which Met80 is displaced from iron by a lysine residue.32,33

In the acid-unfolded form at pH 2, a high-spin electron configuration indicates that neither

Met80 nor His 18 remains coordinated to iron.34,35 At pH 2 in the presence of stabilizing anions, a partially folded form can be obtained.36 In the presence of high concentrations of dénaturant (guanidine hydrochloride or urea), the protein conformation approximates a random-coil polypeptide devoid of a circular dichroism (CD) band at 222 nm. A recent

NMR spectroscopic study of iron cytochrome c in neutral solution detected His and Lys upon partial and His and His axial ligands upon complete denaturation with urea.37 The molten-globule form, designated MG, is a thermodynamically distinct, compact denatured form with a significant amount of ordered secondary structure, but largely flexible, disordered, and fluctuating tertiary structure. In comparison with the native form, the internal hydrophobic core of the protein is more exposed to solvent, and side chains are more mobile.38,39 The MG form preserves many native internal hydration sites and is hydrated on the surface like the native form.40 Several structural studies found a major intermediate in protein folding41-13 that is similar in nature to the MG form.44-18 The MG form has been implicated in various biological processes,49 such as interaction of nascent proteins with molecular chaperones50,51 and interaction of proteins with membranes.52

Cytochrome c undergoes a transition from the acid-unfolded (UaCid) to the molten- globule (MG) form upon addition of anions (as salts).33,38,53™55 At pH 2.0 in the absence of salt, cyt(IH) is maximally unfolded because of electrostatic repulsions within the protein.

Upon addition of salt, that is, at higher ionic strength, the added anions screen the cationic groups, and the protein cooperatively folds into a compact structure. This, the MG form of cyt(IH), has been extensively characterized.56-58 Its a-helicity is comparable to that of the native form, but it has a fluctuating tertiary structure. The hydrophobic core, containing the

N-terminal and C-terminal helicies and the heme group, is preserved in the MG form and still stabilized by nonbonding interactions, while the loop regions are fluctuating and partially disordered.59,60 Of the two axial ligands, only His18 seems to remain attached to iron in the

61 MG form, while Met80 is detached. The radius of gyration of the native, MG, Uacjd, and

Uurea forms of iron cytochrome c increases in the order 14.6 < 17.4 < 30.1 < 32.1 Â, respectively.62 38

The replacement of heme iron by zinc(II) in myoglobin, hemoglobin, cytochrome c

peroxidase, and cytochrome c has successfully been used to probe the structure and reactivity

of these proteins in the folded state.63"71 Spectroscopic studies of folded zinc-substituted

cytochrome c, designated Zncyt, showed that metal substitution does not significantly alter

the protein structure near the heme pocket.72-74 Ours is the first study of unfolded forms of

this protein. We characterize them on the basis of far-UV circular dichroism spectra, Soret

absorption spectra, their behavior upon unfolding by acid and by urea, and rate constant for

natural decay of the triplet state.

Zinc cytochrome c offers many advantages over the native species. The long-lived

triplet state of the zinc porphyrin, designated 3Zncyt, is easily produced by laser flash and is a

strong reductant, suitable for exact kinetic studies. The triplet state is oxidatively quenched

by electron acceptors Q according to eq 1; four such reactions are the subject of this study.

3Zncyt + Q —> Zncyt* + Q~ (1)

The resulting cation radical, designated Zncyt*, returns to the ground state, Zncyt, in the

thermal (so-called back) electron-transfer reaction shown in eq 2. This reaction was the

Zncyt* + Q~ —> Zncyt + Q (2)

subject of a recent study in our laboratory.69

Although overall structure and stability of cytochrome c in unfolded forms has

recently been studied, reactivity of these forms remains almost unknown. We explore the effects of conformational change, by comparing electron-transfer kinetics of 3Zncyt in folded and unfolded forms, in neutral and acidic solutions, and in the presence of urea, a dénaturant.

Because Zncyt and most of the quenchers are charged, we also control the electrostatic 39 interactions between them by using low and high ionic strength.

Experimental Section

Chemicals. Distilled water was demineralized to a resistivity greater than 17

MQecm. Chromatography resins and gels were obtained from Sigma, Pharmacia, and Bio-

Rad. Hydrogen fluoride, nitrogen, and ultrapure argon were obtained from Air Products.

e Tris( 1,10-phenanthroline)cobalt(m) perchlorate dihydrate, [Co(phen)3](C10 4)3 2H20, and the corresponding chloride salt, [Co(phen)3]Cl3'7H,O, were prepared by published procedures.75

(Caution, perchlorate salts of metal complexes containing organic ligands are potentially explosive! Only small amounts of them should be prepared and must be handled with great

3+ caution.) The Co(phen)3 concentration was determined on the basis of the molar absorptivity: e = 3.60 x 103 M"1 cm-1 at 350 nm.76 Tris(acetylacetonato)cobalt(m),

Co(acac)], from Aldrich was 99.99% pure. Its concentration was determined on the basis of the molar absorptivity: e = 133 M"1 cm-1 at 595 nm.77 All other chemicals were of reagent grade, and were used as received from Fisher Scientific.

Proteins. Horse heart cytochrome c was obtained from Sigma. The iron(III) form was prepared by incubation with an excess of K3[Fe(CN)e], which was then removed with a

Bio-Rad Econo-Pac 10 DG desalting column. The protein concentration was determined spectrophotometrically; the difference in molar absorptivity between reduced and oxidized forms at 550 nm is 18.5 mM™1 cm"1.

Zinc cytochrome c was prepared and purified by modification70,74 of the original procedure.78,79 All experiments were done in the dark, to prevent photodegradation of Zncyt in the presence of dioxygen. Two of the criteria of purity were the absorbance ratios

A423/A549 > 15.4 and A549/A585 < 2.0. Another criterion of purity was that the rate constant &d

for the natural decay of 3Zncyt be less than 110 s-1. The concentration of Zncyt was

determined on the basis of its molar absorptivity: e = 243 mM-1 cm-1 at 423 nm.80 The

metal-free form of cytochrome c is porphyrin cytochrome c, designated H2cyt. It was

prepared by a standard procedure.78

Preparation of Buffer and Dénaturant Solutions. The phosphate buffer had a

concentration of 10 mM and pH value of 7.00 ± 0.05, determined by an Accumet 925 pH

meter obtained from Fisher Scientific. Aqueous solutions of HC1 were adjusted to pH 2.0.

The ionic strength, |n, of the buffer at pH 7.00 or of the HC1 solution at pH 2.0 was adjusted

to 20 mM and 1.500 M by addition of solid NaCl. Solutions of 8.0 M urea in the 10 mM

phosphate buffer at pH 7.00 and g = 20 mM were prepared fresh by a published procedure81

and used within 8 h. Buffers and dénaturant solutions were thoroughly degassed before use

by bubbling with wet argon.

Preparation of (Un)folded Forms of Zncyt. The folded forms at low and high ionic

strength are designated Fiow and Fhigh- They were obtained by dissolving Zncyt in 10 mM

phosphate buffer at pH 7.00 and ionic strength of 20 mM and 1.500 M. The acid-unfolded form, designated UaCjd, was obtained by dissolving Zncyt in the HC1 solution at pH 2.0 and n

= 20 mM. To obtain the molten-globule form, designated MG, the aforementioned solution was adjusted to 1.500 M by addition of solid NaCl, and pH was readjusted to 2.0 by HCI.

Precautions were taken to avoid pH dropping below 2.0 lest zinc(H) ions irreversibly dissociate from porphyrin. This undesirable process can be detected, and thus avoided, 41

because the absorption spectrum is characteristic of H%cyt. The urea-unfolded form,

designated Uurea, was obtained by dissolving Zncyt in a solution of 8.0 M urea in 10 mM

phosphate buffer at pH 7.00 and ji = 20 mM. Transitions between the conformational forms

were induced by acid-base titration, NaCl addition, or solvent exchange with Centricon-3

ultrafiltration units, obtained from Millipore.

Circular Dichroism and Absorption Spectrophotometry. Far-UV circular

dichroism spectra of 10.0 pM Zncyt were obtained with a Jasco instrument, Model J-710. A

1-mm quartz cell was used for the Ftow, Fhjgh, MG, and Uacid forms, but owing to the strong

absorbance of urea, a 0.1-mm quartz cell was necessary for the Uurea form. Each spectrum

was an average of four measurements and was slightly corrected for a baseline contribution.

The cell compartment was maintained at 20.0 ±0.1 °C using a peltier thermoelectric cell

PTC-348 W, equipped with an external sensor. The mean residue ellipticity, in deg cm2

-1 dmol , is defined as [5] = lOO0obs(c/r\ where 0„bS is observed intensity, c is concentration in

residue moles per liter, and I is path length in centimeters. Helical content was calculated by

an established method.82

Absorption spectra of Zncyt in the Fiow, Fhigh, MG, Uacid, and Uurea forms were

recorded with a Perkin-EImer Lambda 18 spectrophotometer. To determine the stability of

protein toward demetalation, 5.0 fiM solutions of Zncyt or cyt(HI) in solvents maintaining all

five folded and unfolded forms were monitored over time.

Equilibrium Unfolding of Zncyt. Controlled denaturation of Zncyt was monitored

by Soret absorption at 410 and 423 nm, at various concentrations of HC1 (pH 1.1-7.0) and of urea (0.0-12.0 M). The unfolding transition at 20 °C was analyzed in terms of a two-state 42

model, and the optical data were converted into the free energy of unfolding (AGu) using eq

3, where y, yp. and _yu are the observed spectroscopic signal, folded-protein baseline, and

\ (3)

unfolded-protein baseline, respectively. The midpoint of the unfolding transition (Cm) is

obtained by fitting a four-parameter sigmoidal equation (SigmaPlot version 5.0, from Jandel

Scientific) to the fraction of unfolded protein (Fu) and dénaturant concentration. Linear

least-squares regression was used to fit the data to eq 4, where AGu(HiO) is an estimate of

AGu = AGu(H%0) - ^[dénaturant] (4)

the free energy of unfolding in aqueous solution, and m is a measure of the dependence of

81,83 AGU(H20) on dénaturant.

Laser Flash Photolysis. Transient absorption of the triplet state, 3Zncyt, as a

function of time was measured with a standard apparatus. The excitation source was a

Continuum Minilite H Q-switched frequency-doubled Nd:YAG laser, which delivered 5-ns

pulses at 532 nm. Incident energy was calibrated with a power meter (Scientech, model

H410D) and kept at 1.0 mJ/pulse, to avoid Zncyt degradation. The probe source,

perpendicular to the excitation beam, was a continuous 250 W QTH lamp with Aspherab condenser (Oriel Instruments, model 66198). A secondary lens focused the collimated light on a 1-mm aperature. Interference filters (Optometries USA, half-bandwidth of ± 5 nm) placed before the sample holder and also before the detector minimized the entry of scattered light into the detector. Decay of the triplet state, 3Zncyt, was monitored at 458 nm, where its

(transient) absorbance reaches a maximum. The appearance and disappearance of the 43

porphyrin cation radical, Zncyt+, was monitored at 676 nm, where the difference in

absorbance between Zncyt+ and 3Zncyt is the greatest.80,84,85 Transient signals shorter than 1

|is were measured with a Hamamatsu R2949 photomultiplier tube equipped with a housing

and voltage divider (Oriel Instruments, model 70680). The current was amplified (Oriel

Instruments, model 70223) and digitized using a Lecroy Waverunner LT322 oscilloscope

interfaced to a computer. Between 50 and 300 laser shots were accumulated, to enhance the

signal. Transient signals longer than 1 (is were measured with a custom-built photodiode

detector containing a Hamamatsu silicon PIN photodiode model S3071. The photodiode

detector provided greater sensitivity and better signal-to-noise ratio than the photomultiplier

tube did, for observing the small changes in absorbance of the transient species 3Zncyt and

Zncyt+.

A 2.000-mL sample solution in a 10-mm cuvette was thoroughly deaerated by gentle

flushing with wet argon for at least 15 minutes after each addition of quencher or until the

rate constant for natural decay of 3Zncyt became constant. For experiments at pH 7.00, a

deoxygenating solution containing glucose, glucose oxidase, and catalase was used, to ensure

complete removal of dioxygen.80 Temperature was held at 25.0 ±0.1 °C by a thermostated

bath CH/P 2067, obtained from Forma Scientific.

The concentration of Zncyt was 3.0 (JM in experiments done at pH 7.00 and 5.0 PM in those done at pH 2.0 or in the presence of urea. The concentration of the triplet state,

3Zncyt, depended on the excitation power and was ca. 0.3 to 0.5 (jM. The mole ratio of quencher to 3Zncyt was greater than 10:1, so that the conditions for pseudo-first-order reaction always prevailed. 44

Reaction Mechanism. A general mechanism for the reaction between electron donor

D and the electron acceptor A (the same as Q in eq 1) is shown in eq 5. The quotient kon/koff

D+A •DA —» D+ A" (5) *off is the association constant KA and k{ is the rate constant (for the reaction in the "forward" direction). Under the so-called improved steady-state approximation and the condition [A]

» [D], eq 6 is obtained. In the limiting case when kof{ > &<,„[A] + k{, eq 6 yields eq 7. In this •--oSer

*obs - [A] = *bim[A] (7)

case, common in studies of protein reactions, the observed rate constant k0bS linearly depends

on the concentration of the reactant present in excess. The bimolecular rate constant yfcbim corresponds to the reactions of 3Zncyt with various quenchers A.

Fittings of Kinetic Data. The rate constants were obtained from changes in the absorbance at 458 and 676 nm with time. Exponential decay of 3Zncyt in the absence and presence of quencher was fitted using nonlinear least-squares regression (SigmaPlot, version

5.0, from Jandel Scientific). In the absence of quencher, the rate constant for natural decay is designated kd. In the presence of quencher, the quenching rate constant kq is obtained using eq 8. Plots of kq against quencher concentration are linear and go through the origin. The

*q=*obs"*d (8)

bimolecular rate constant kbm is determined from the slope by linear least-squares regression. 45

Results

Characterization of (Un)folded Forms of Zncyt by Absorption Spectroscopy.

Figure 1 and Table 1 show the absorption spectra of Zncyt in the Fiow, Fhjgh, MG, Uacid, and

Uurea forms. Variation in the absorption is diagnostic of changes in conformation. Upon

conversion of Zncyt from the Fiow and Ffogh forms to the MG form, the Soret and Q bands

move by only 1-3 nm. Upon unfolding of Zncyt to the UaCjd form, the Soret band moves

greatly, by 14 nm. The blue shift of the Q band is consistent with a change in zinc(II)

ligation, an increase in the volume of the hydrophobic core, and an increase in porphyrin

exposure to solvent. Upon unfolding of Zncyt to the Uurea form, the blue shift of the Soret

maximum is less than that in the UaCid form. Soret absorption is the only optical property

which can clearly distinguish the folded and unfolded forms of Zncyt.

Equilibrium Unfolding of Zncyt. Figure 2 and Table 2 describe unfolding of Zncyt.

The protein is rather stable, with the midpoint of the unfolding transition (Cm) at pH 2.9 ±0.1

with acid and at 7.1 ±0.1 M with urea as a dénaturant. As shown in Figure 2a, unfolded

Zncyt refolds upon lowering pH below 2.0 with HC1. This refolding, having Cm at pH 1.5 ±

0.2, arises from the increase in the concentration of CI" ions, which induce conversion of

Zncyt from the Uacid form to the MG form. The unfolding curves in this study of Zncyt, and

38 54 57 83 those in previous studies of iron cytochrome C) - - - confirm that the Uacid and Uurca forms

of both proteins are completely unfolded at the conditions used in our kinetic studies, namely

pH 2.0 and 8.0 M urea.

Characterization of (Un)folded Forms of Zncyt by Circular Dichroism

Spectroscopy. Circular dichroism in the far-UV range is a property of the polypeptide 46

backbone and is a sensitive indicator of protein secondary structure. Circular dichroism

spectra (Figure 3 and Table 1) of Zncyt in the Ftow, F^gh, and MG forms show minima at 222

and 210 nm and have similar a-helical content, indicating similar backbone conformations.

These are the same as those recently reported74,86 and also very similar to those reported for

native (iron-containing) cyt(m).54 By contrast, the spectra of Zncyt in the Uacjd and Uurea

forms show minima below 210 nm and a great decrease in ellipticity at 222 nm. In lacking

secondary structure, these forms resemble the corresponding unfolded forms of cyt(III)38,54

and Zncyt86 previously reported.

Characterization of (Un)folded Forms of Zncyt by Natural Decay of the Triplet

State. Natural decay in the absence of quenchers, shown in Figure 4, usually is the simple

process shown in eq 9 that obeys the monoexponential eq 10. When the MG and Uacid forms

3Zncyt —> Zncyt (9)

M,58nm = exp(-*df )+b (10) of Zncyt are left to age for several hours, a second phase in the natural decay arises gradually, so that the biexponential eq 11 is needed for the fitting. The new phase is faster but always

= a Missnm \ exp(- kxt )+ a2 exp(- k2t )+b (11) remains a minor one.

Its rate constants (fe) and amplitudes (a?) are 710 ± 90 s™1 and 10 to 20% for the MG

-1 form and 1230 ± 250 s and 20 to 30% for the Uacid form. An extreme example of this

3 biphasic natural decay is shown in Figure 5. Natural decay of the Uurea form of Zncyt is always biexponential. For the major phase, k\ = 1200 ± 220 s_1 and a\ = 82 ± 6%; for the

-1 minor phase, k2 = 280 ± 80 s and <22 = 18 ± 6%. 47

The k

Conformational Transitions Between (Un)folded Forms of Zncyt. Various forms of Zncyt were converted one into another as the solvents (specified previously for each conformational form) were exchanged by ultrafiltration. The interconversions were followed on the basis of the absorption and CD spectra and the natural decay of the triplet state.

Gradual conversion of the Fhjgh form into the MG form and the F|OW form into the UaCjd form was followed over 36 h. Attempts to reverse these processes and gradually, again over

36 h, reform the F^gh form from the MG form and the Ftow form from the UaCjd form failed.

The Soret bands of these folded forms and emission of the triplet state did not reappear.

Instead, absorption spectra with bands at 404, 506, 540, 568, and 620 nm were obtained (see

Figure S1 in the Supporting Information). These bands are characteristic of the metal-free porphyrin cytochrome c, designated Hzcyt.78 Circular dichroism spectra shown in Figure S2 in the Supporting Information showed recovery of nativelike secondary structure in the Fhigh and Fiow forms of Hicyt. When, however, ultrafiltration was done rapidly, the MG form did

return to the Fhjgh form and the UaCjd form to the Ftow form, as the position of the Soret bands

1 (at 423 nm) and the rate constants kd for natural decay (ca. 75 s™ ) showed.

Conversion of the UaCid form into the MG form was effected by adding NaCl to the solution and monitored by measuring ellipticity [6\ at 222 nm and absorbance changes in the Soret region. As the ionic strength is raised from 20 to 1000 mM, Zncyt recovers nativelike

secondary structure (data not shown). As Figure S3 in the Supporting Information shows,

isosbestic points at 398 and 417 nm persist as the ionic strength is raised from 20 to 110 mM,

evidence for a single process. These isosbestic points become somewhat "smeared" upon

going to 1000 mM, presumably because of processes occurring simultaneously.

Demelalation of Zncyt and Remetalation of H2cyt by Zinc(II) Ions. When the

MG and UaCid forms of Zncyt are left to age for several hours, a second, faster phase in the

natural decay arises gradually. To attribute this faster phase, the natural decay of the triplet

3 state of the metal-free porphyrin cytochrome c, designated H2cyt, in the absence of

1 quenchers was measured (see Table 1). The value of kd (1350 ± 70 s" ) determined for this

_l H2cyt sample is identical to the value of fc2 (1353 ± 30 s ) determined for the aged Zncyt sample in Figure 5.

To check whether the observed biphasic kinetics at pH 2.0 is indeed due to an increase in H2cyt concentration as the zinc(H) porphyrin is demetalated and to ensure that the

3 appearance of H2cyt will have no effect on the rate constant for natural decay of Zncyt, increasing concentrations of H2cyt were added to a 10.0 |iM solution of Zncyt for determination of kd. Fitting of the kinetic traces to eq 11 yielded the rate constants t, and ki and the amplitudes a\ and a2 for the two phases. A plot of kq versus increasing concentration

1 of H2cyt (data not shown) shows a very slight dependence of (slope = 0.23, 79 s™

1 -1 105 s" ) and no dependence of kj (slope = 0.002, fc2 = 1440 ± 50 s ) on H2cyt concentration.

Figure 6 shows that the faster phase, which arises over several hours, is indeed due to formation of H2cyt (see Scheme 1). The molecules Zncyt and H2cyt quench each other's 49 triplet states slightly, if at all. Indeed, lack of a transient signal at 676 nm rules out the presence of Zncyt\ the intermediate that would have arisen in redox quenching of 3Zncyt by

Hicyt. The slight quenching observed can be attributed to slight energy transfer.70

Our attempts to suppress loss of zinc(II) ions from Zncyt, that is, to remetalate H2cyt in situ by adding 1.0 mM zinc(II) acetate to the reaction mixture at pH 2.0 failed. See, however, Figure S4 in the Supporting Information for successful remetalation of Hicyt using standard procedures.74'78 Restoration of Zncyt was confirmed by the position of the Soret

-1 band (at 423 nm) and the rate constant kd for natural decay (72 s ).

Effect of Zncyt Conformation on the Porphyrin Affinity for Zinc(II) Ions. To

understand the stability toward demetalation of Zncyt and cyt(III) in the Fhjgh, Ftow, MG, UaCjd, and Uurea forms, changes in their absorption spectra over time were monitored. As Figure 7

shows, the rate of demetalation at pH 2.0 is greatest when Zncyt adopts the UaCid form. Lack

of change in the absorbance for the Uurea form (see Figure 7d) suggests that demetalation of

Zncyt requires acidic conditions, presumably because hydrogen ions assist displacement of zinc(II) ions at pH 2.0 (see Scheme 1). No change in absorbance was observed for Zncyt in

the Fhigh form after 7 days at pH 7.00 and 4 °C (data not shown) and for cyt(IH) in the UaCid and MG forms after 2 and 5 days, respectively (see Figure S5 in the Supporting Information).

Most importantly, these results show that flash photolysis experiments can be performed before loss of zinc(H) ions progresses enough to complicate analysis of the kinetics. By doing the experiments with fresh samples, we avoided these complications.

Oxidative Quenching of 3Zncyt by Inorganic Complexes and Cyt(III). To test the effect of the protein conformation on reactivity, we compared the electron-transfer properties 50

3 of Zncyt in the Flow, Fhigh, MG, Uacid, and Uurea forms. The probes of Zncyt reactivity were

3 the following three inorganic and one protein electron acceptors: anionic Fe(CN)6 ~, cationic

Co(phen>33+, electroneutral Co(acac)3, and cationic cyt(IH). This native protein was chosen

as a probe since both it and its zinc derivative will be subject to similar unfolding. Ionic

strength and pH for obtaining the five folded and unfolded forms of Zncyt are chosen in some

cases to bring out, but in most cases to minimize, effects of electrostatic interactions between

this protein and the oxidizing agents, so that effects of conformation on reactivity become discernible. The results are shown in Figure 8.

In 18 out of 20 cases, presence of quenchers significantly increases the decay of the triplet state 3Zncyt. Only slight, but reproducible, increases occur upon addition of

Co(phen)33+ and cyt(IH) at low ionic strength. Evidently, electrostatic repulsions limit collisional quenching between two cationic species: the Fiow form of Zncyt and the quencher.

When ionic strength is raised to 1.500 M, electrostatic interactions are suppressed, and

3 diffusional quenching occurs. In the Ftow and Fhigh forms, decay of the triplet state, Zncyt,

remained monoexponential upon addition of all four quenchers. In the MG, UaCid, and Uurea forms, however, the decay was biexponential. Typically, the faster phase accounted for only

ca. 15% - 20% of the total amplitude. In the Uurea form, a slower phase accounted for only

10% - 15% of the total amplitude. Because these minor phases parallel the major ones in reactivity (upon addition of quenchers to different conformational forms of 3Zncyt) we will focus on the major phases in each reaction. The psuedo-first-order rate constants are directly proportional to quencher concentration and depend on ionic strength and conformation of

Zncyt, as Figure 8 shows. The second-order rate constants, obtained from the slopes of the 51 linear plots, are listed in Table 3.

Discussion

Structure and Reactivity of (Un)folded Forms of Cyt(III) and Zncyt. The conformation of iron cytochrome c as a function of temperature, pH, dénaturant, and salt concentration has been well studied. Recent investigations suggested that folded metal- substituted cytochrome c and iron cytochrome c have similar tertiary structures,72-74'83 but partially and completely unfolded forms of Zncyt have not yet been fully characterized.87 We characterize them in this study. Our goal is not to compare zinc and iron forms of cytochrome c, but to study electron-transfer reactivity of Zncyt in various conformational forms. Ours is the first study of its kind.

The reduction potential of heme proteins is influenced by identity, alignment, and basicity of the axial ligands; distortions of the heme; and local charges. Heme exposure to solvent is particularly important; decrease in this exposure correlates with an increase in

88 reduction potential. Reduction potentials of the Fhigh (+255 mV) and MG (+233 mV) forms of cyt(ni) are similar,89 but that of the forms unfolded by guanidine hydrochloride90 or urea91

(ca. -150 mV) is much lower and close to the potential (ca. -200 mV) of bis(imidazole) iron(m) porphyrin in aqueous solution.92™94 (All the potentials are given versus NHE.)

Absorption and Circular Dichroism Spectra of (Un)folded Forms of Zncyt. The axial coordination to the zinc(II) ion in Zncyt and to the iron ion in unfolded forms of iron cytochrome c is still uncertain. A recent study suggested that zinc(II) is six-coordinate (with

His18 and Met80 ligation) in the Fiow (pH 5, low salt concentration) form and five-coordinate 86 74 (with MetSO detached) in the MG (pH 2.0, 1.00 M NaC104) form. Reconsideration of

73 NMR spectra suggested that even the F|0W form of Zncyt is five-coordinate, that is, that

MetSO is not coordinated. Similarity of the absorption spectra for the Ftow and MG forms of

Zncyt (see Table 1 and Figure 1) suggests that these forms have similar ligation. Complete unfolding of Zncyt by acid or urea induces large changes in absorbance, presumably owing to changes in zinc(II) ligation and increases in porphyrin exposure to solvent. Interestingly,

absorption spectra of our Zncyt in the UaCjd form and of the Zncyt in the MG form induced by

86 - C104~ are similar. In cyt(IH), small anions (such as Cl~, Br", and NO3 ) induce formation of a compact, highly structured MG form, restoring the native Fe(HI)-Met80 bond and nativelike redox properties. Large anions (such as I~, C104~, and CCI3COO") induce formation of a compact MG form that has bis-His coordination to heme iron and a fluctuating tertiary structure and lacks nativelike redox properties. Chloride and C104"" anions stabilize

MG forms of iron cytochrome c having nativelike a-helical structures, but having distinct tertiary conformations in which highly flexible loop regions are responsible for different spectroscopic and redox properties.89 Our results conclusively show that the MG form of

Zncyt can be achieved, although it is unclear whether the axial ligands in it are the same as those in the MG form of iron cytochrome c.

Our experiments with acid and urea consistently show that identity of the metal ion in cytochrome c does not significantly affect the protein behavior in the completely unfolded

(Uacid and Uurea) forms. The metal ion, its oxidation state, and its axial ligand(s) in cytochrome c affect the free energy of unfolding in aqueous solution, AGu(H20).

Consequently, equilibrium unfolding of iron cytochrome c induced by guanidine 53 hydrochloride shows that cyt(II) is 42 kJ mol-1 more stable than cyt(IH) towards unfolding.95

Values of AGu(HiO) estimated by urea-induced unfolding follow the trend cyt(H) > Zncyt =

96 cyt(III) » H2cyt. We chose the conditions for the UaCjd and Uurea forms, to ensure that

Zncyt and iron cytochrome c behave the same under extreme conditions (pH 2.0 or 8.0 M urea). Most important, the biphasic kinetics observed in aged Zncyt samples cannot be attributed to heterogeneity due to incomplete unfolding of Zncyt.

Far-UV circular dichroism (see Figure 3) supports the conclusion that Zncyt and iron

54,72,74,86 cytochrome c in the F|0W, Fhigh, and MG forms have almost the same conformation.

Marked loss of ellipticity at 222 nm for Zncyt in the Uacjd and Uurea forms indicates lack of

38,54 secondary structure, as with iron cytochrome c in UaCjd and Uurea forms.

Natural Decay of the Triplet State of Zncyt in Different (Un)folded Forms. Table

3 1 shows that the rate constant kd for natural decay of Zncyt increases in the order Fhigh < F|OW

< MG < UaCjd < Uurea- Independence of this rate constant on protein concentration confirms that unfolding is an intramolecular process. Excited-state energies of metalloporphyrins are strongly affected by axial ligation and solvent.97 The internal hydrophobic core in the MG form expands somewhat and becomes more exposed to solvent, hence, a slight increase in the rate constant for natural decay. Upon complete unfolding of Zncyt by acid or urea, and probable displacement of His 18 and MetSO by solvent,34 35 this rate constant increases

3 greatly. Because the rate constants for natural decay of Zncyt in the UaCjd and Uurea forms are different, our use of two denaturing agents is justified. These results support the conclusion

that in Zncyt the change in the rate constant for natural decay between the Ftow, Fhigh, MG,

UaCid, and Uurea forms is due to differences in zinc(II) ligation, porphyrin accessibility to 54

solvent, and energy of the triplet state.

Reactivity of 3Zncyt in the Five (Un)foided Forms. Metal complexes, with their

well-defined structural and chemical properties, are well suited as probes of the degree of

Zncyt unfolding. Because cytochrome c uses essentially the same surface patches for

interactions with "small" reactants and with proteins, the former have often been used as

substitutes for the latter in informative studies.9 The reaction in eq 1 is bimolecular with all

quenchers and under all conditions studied. The linear plots in Figure 8 indicate a simple

collisional mechanism of quenching, which does not require the involvement of a persistent

protein-quencher complex.

A) Role of Electrostatic Interactions. Electrostatic interactions have a dominant

3 effect on Zncyt reactivity. Assuming normal pATa values, Zncyt and cyt(IH) at pH 7.0 have

overall charges of +6 and +7, respectively. At low ionic strength (20 mM), electrostatic

repulsion between Zncyt on one side and Co(phen)33+ or cyt(IE) on the other hinders their

3+ association. The very slight dependence of kq on concentration of Co(phen)3 and cyt(III) is

evident in the very small values of &bjm in Table 3. At high ionic strength (1.500 M) the two

cationic species overcome the repulsions and interact; the reactivity of 3Zncyt with

Co(phen)33+ and with cyt(III) increases 245-fold and 7.0-fold, respectively. Reaction between

3 3 oppositely charged partners Zncyt and Fe(CN)6 ~ is virtually diffusion-controlled at the low

ionic strength, and is slowed down 20-fold at the high ionic strength.

3 Comparison of Zncyt reactivity in the MG (p. = 1500 mM) and UaCjd (|x = 20 mM) forms is only tentative because the effects of electrostatic interactions and porphyrin exposure

3+ may not be separable. In the case of Co(phen)3 , the rate constant Jtbim decreases 2.0-fold as 55

Zncyt changes from the MG form to the Uacid form, presumably because the highly

protonated latter form has a greater positive charge than the former form. In the case of

cyt(m), however, kbim increases 3.6-fold from the MG form to the Uacjd form despite the

enhanced electrostatic repulsion. The increased reactivity presumably is caused by a greater

exposure of porphyrin in both Zncyt and cyt(m) upon unfolding.

To remove the ambiguities caused by electrostatic interactions and bring out the

effects of (un)folding on reactivity, we chose the electroneutral oxidant Co(acac)]. It shows

3 the same rate constant for quenching the Ftow and Fhigh forms of Zncyt; indeed, this reaction

3 is independent of ionic strength. Reactivity of Zncyt in the Fhjgh form (under conditions that

suppress electrostatic interactions) with FetCN^3-, Co(phen)33+, and Co(acac)3 follows the

decrease in the reduction potential of the inorganic quencher, respectively, in the order +410,

+370, and -340 mV.

B) Partial Unfolding of Zncyt: The Molten-Globule Form. Although the MG form of Zncyt is extensively protonated, at high ionic strength the intramolecular electrostatic repulsions are suppressed, allowing the protein to maintain a compact, nativelike conformation. Direct comparisons between the F^gh and MG forms of Zncyt, without complications from electrostatic interactions, are possible. In going from the F^ form to the

MG form, the rate constant Xtim increases 1.4-fold, 1.6-fold, 4.0-fold, and 10-fold in the order

3- 3+ cyt(m), FetCN^ , Co(phen)3 , and Co(acac)3. The increase is slight for the first two quenchers but larger for the last two, presumably because of their different surface characteristics. The basic recognition patch of cytochrome c and the cyano complexes are hydrophilic, whereas the phen and acac complexes are hydrophobic. The last two quenchers 56

can better take advantage of the enhanced hydrophobicity caused by the partial exposure of

porphyrin in the MG form.

C) Complete Unfolding of Zncyt: The UaCid and Uurea Forms. Because both of

these unfolded forms of Zncyt exist at low ionic strength (20 mM), they can be properly

3 compared with the folded Ftow form. Reactivity toward the anionic quencher Fe(CN)6 ~ is

slightly lowered upon unfolding of the Ftow form. The kbim values for the Uacid and Uurea

forms are virtually the same. Because these three forms of 3Zncyt react with Fe(CN)63~ at

essentially diffusion-controlled rates, these reactions are least informative about the effects of

unfolding.

Reactivity toward the cationic quenchers cyt(III) and Co(phen)33+ is enhanced more

when the unfolding agent is urea (168-fold and 1340-fold, respectively) than when this agent is acid (36-fold and 466-fold, respectively) because Zncyt has a lower positive charge in the

Uurea form than in the UaCjd form. With either method of unfolding, the hydrophobic

Co(phen)33+ benefits more than the hydrophilic cyt(HT) from the greater accessibility of the hydrophobic porphyrin cofactor in Zncyt. The metal complex, being relatively small, also benefits more than cyt(III) does from the greater exposure of porphyrin in Zncyt.

The electroneutral complex Co(acac)3 reacts at the same rate with the UaCid and U^ea forms of 3Zncyt. In other words, unfolding enhances the quenching reaction to virtually the same extent (62-fold and 54-fold, respectively) regardless of the agent used for the unfolding.

When electrostatic interactions are eliminated, the effects on the reactivity of increased exposure of the porphyrin cofactor and of the closer approach by the quencher can be assessed. 57

Conclusions

Electron-transfer reactivity of the triplet state of zinc-substituted cytochrome c in the

folded (at low and high ionic strength), molten-globule, and unfolded (by acid and urea)

forms is probed with four different quenchers. This reactivity depends mostly on electrostatic

interactions and the degree of porphyrin exposure. Using the electroneutral complex

Co(acac)3 as a quencher, we eliminated the electrostatic effects and assessed the

consequences of porphyrin exposure for the electron-transfer reactivity. The rate constant for

the photoinduced reaction in eq 1 increases 10-fold upon partial unfolding into the molten-

globule form and again approximately 5-fold upon further, complete unfolding of zinc

cytochrome c.

Acknowledgment. We thank Dr. Tonu Reinot for many discussions on transient

absorption spectroscopy. This study was supported by the U. S. National Science Foundation

through Grant MCB-9808392.

Supporting Information Available: Five figures showing the absorption and far-

UV circular dichroism spectra of Zncyt and H%cyt illustrating Zncyt demetalation; change in

absorption spectrum of Zncyt in the UaCjd form upon addition of NaCl stabilizing the MG

form; change in absorption spectrum upon remetalation of H%cyt forming Zncyt; and stability

of cyt(m) in the MG and UaCjd form (5 pages). This material is available free of charge via

the Internet at http://pubs.acs.org.

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Tables and Figures

Table 1. Characterization of the Folded Forms (at Low and High Ionic Strength), Molten-

Globule Form, and Forms Unfolded by Acid and by Urea of Zinc Cytochrome c and Metal-

Free Porphyrin Cytochrome c at 25 °C and Different Values of pH and Ionic Strength (|U.)

molar elliptic!ty absorption maxima. À™ (nm) a-helical ~{0\ 2^ natural decay Soret content protein form' PH H (mM) band Qbands (deg cm" dmol ') <%) *d(S~')

Zncyt flow 7.00 20 423 549 585 10300 28.0 70 ±4

Rugb 7.00 1500 423 549 585 11150 31.6 63 ±3

MG 2.0 1500 421 548 582 10530 28.1 190*20

Uacid 2.0 20 407 547 575 4120 16.4 440 ±50

7.00 20 418 546 581 1880 4.4 1200 ± 220

H;cyt F^ugh 7.00 1500 404 506 540 568 620 11070 32.6 1350 ±70

MG 2.0 1500 405 506 538 559

Uacid 2.0 20 406 552 594

Uurea 7.00 20 400 504 540 565 616

3 Ftow and Fhigh, folded forms at low and high ionic strength; MG, molten-globule form; and

Uacid and Uurea, forms unfolded by acid and by urea. 65

Table 2. Thermodynamic Parameters for Urea-Induced Unfolding of Zinc Cytochrome c,

Iron(III) Cytochrome c, and Metal-Free Porphyrin Cytochrome c at 20 °C

AG u(H20) m cm protein (kJ mol"1) (kJ mol"' M~') (M) Zncyt1 47.2 ±3.3 6.6 ±0.5 7.1 ±0.1

cyt(ffl)b 37.5 5.0 7.5

b H:cyt 6.1 3.6 1.7 1 This work.

b From ref 83; estimated errors are, at most, ±10%.

Table 3. Bimolecular Rate Constants for Reactions of the Triplet State of Zinc Cytochrome

c in Different Conformational Forms with the Four Oxidative Quenchers Shown, at Different

Values of pH and Ionic Strength (p,)

conditions A-bim X 10"6, M 1 s~[ at 25 °C

1 3 form PH U(mM) Fe(CN)t> Co(phen)3 * Co(acac)3 cyt(III)

Flow 7.00 20 10700 ± 100 0.44 ± 0.03 3.7 ±0.1 0.56 + 0.07

Fhigh 7.00 1500 543+ 11 108 + 4 3.8 ±0.1 3.9 + 0.2

MG 2.0 1500 882 ± 17 443+ 11 39 ± I 5.5 ± 0.4

Uacid 2.0 20 8020 + 590 205 ±2 230 ±11 20 ± 1

Uum 7.00 20 6260 + 350 591 ±5 200 ±9 94 ±11 a Fiow and Fhigh, folded forms at low and high ionic strength; MG, molten-globule form; and

UaCid and Uurea, forms unfolded by acid and by urea. + 2+ 2H +Zncyt —H2cyt+ Zn

Zncyt ———> 3Zncyt —^slow^—> Zncyt

3 H2cyt ———> H2cyt ——> H2cyt

Scheme 1 67

add

high 8 c 1 1 < 0.4 -

0.2 -

0.0 360 380 400 420 440 460 Wavelength (nm)

0.10

high U«cld 0.08 - MG

urea 3 c 0.06 - 1

ja o.04 -

0.02 -

0.00 480 500 520 540 560 580 600 620 640 Wavelength (nm)

Figure 1. Absorption spectra in the (a) Soret region and (b) Q-band region of the folded form at high ionic strength (Fhigh), molten-globule (MG) form, and the forms unfolded by acid (UaCid) and by urea (Uurea) of 3.0 jjM zinc cytochrome c. The absorption spectrum of the folded form at low ionic strength (F|0W) of zinc cytochrome c (not shown) is identical to that of the Fhigh form. Conditions: F|0W, at pH 7.00 and n = 20 mM; Fhigh, at pH 7.00 and p. = 1.500 M; MG, at pH 2.0 and n = 1.500 M; Uacid, at pH 2.0 and g = 20 mM; and U^ea, at pH 7.00, n = 20 mM, and 8.0 M urea. 68

0 1 2 3 4 5 6 7 pH

0.8 -

0.6 - Uf 0.4 -

0.2 -

0.0 -

0 2 4 6 8 10 12 [Urea] (M)

Figure 2. (a) Equilibrium unfolding of 5.0 jiM zinc cytochrome c at 20 °C, induced by HC1 and monitored by Soret absorption, at 410 nm. Unbuffered aqueous protein solutions with pH adjusted from 7.0 to 1.1 are prepared by addition of dilute HC1 or NaOH. The line is a fitting to four-parameter sigmoidal equations with the midpoint of the unfolding transition (Cm) at pH 2.9 ±0.1 and the midpoint of the refolding transition (Cm) at pH 1.5 ± 0.2. (b) Equilibrium unfolding of 1.0 jiM zinc cytochrome c at 20 °C, induced by urea and monitored by Soret absorption, at 423 nm. Urea concentrations from 0.0 to 12.0 M are prepared by dilution of 12.0 M urea into 10 mM phosphate buffer with pH 7.00 and = 20 mM. The line is a fitting to a four-parameter sigmoidal equation with the midpoint of the unfolding transition (Cm) at 7.1 ±0.1 M. The fraction of unfolded protein (Fu) and free energy for unfolding (AGu) are calculated using eq 3. See Table 2 for the resulting thermodynamic parameters. 69

O E •o 1eo O) 0) B CO urea X

190 200 210 220 230 240 250 260 Wavelength (nm)

Figure 3. Circular dichroism spectra in the far-UV region of the folded forms at low (Fiow) and high (Fhigh) ionic strength, molten-globule (MG) form, and the forms unfolded by acid (Uacid) and urea (Uurea) of 10.0 pM zinc cytochrome c. See Figure 1 for experimental conditions. 70

high ^low MG 0.8 E c S < 0.6 "O N (0 0.4 E ok. z 0.2

0.0

0 2 4 6 8 10 Time (ms)

Figure 4. Natural decay (in the absence of quencher) of the triplet states of zinc cytochrome c (designated 3Zncyt, five traces) and metal-free porphyrin cytochrome c (designated ^Hicyt, one trace), in the folded forms at low (Ftow) and high (Fhigh) ionic strength, molten-globule

(MG) form, and the forms unfolded by acid (Uacjd) and urea (Uurea) of 10.0 |iM zinc cytochrome c or 40.0 |iM metal-free porphyrin cytochrome c. The lines are fittings to eq 10. The upper traces, truncated to the timescale of the figure, continue to decline exponentially. See Figure 1 for experimental conditions. 71

10 15 20 25 30 35 Time (ms)

Figure 5. Natural decay (in the absence of quencher) of the triplet state of zinc cytochrome c that was left for several hours in the UaCid form (at pH 2.0 and p. = 20 mM) before being returned by ultrafiltration to pH 7.00 and |A = 20 mM. In this extreme case of the aged -1 protein sample, fittings to eq 11 gave k\ = 86 ± 3 s~\ a, = 48.5 ± 1.1%, &2 = 1350 ± 30 s , and Û2 = 51.5 ± 0.8%. Quality of the fitting is shown in the plot of the residuals below. 72

1.6

0.8

0.6 CM 0.4

0.2

0.0 0 2 4 6 8 10 12 14 16

[H2cyt] / [Zncyt]

Figure 6. Ratio of amplitudes ai (for the fast phase) and a\ (for the slow phase) depends on the ratio of molar concentrations of the two sensitizers. Changes of transient absorbance with time upon laser excitation of solutions containing 10.0 |iM zinc cytochrome c (Zncyt) and increasing concentrations of metal-free porphyrin cytochrome c (H^cyt) are fitted to eq 11. Invisible error bars are smaller than the symbol. The solvent was 10 mM sodium phosphate buffer at pH 7.00 and n = 1.500 M. 73

- 0.14

0 - 0.12 1 - 0.10 - 0.08

I - 0.06

s I I

s 1

- 0.10 o s - 0 06 U i

i - 0.02

- 0.00

360 380 400 420 440 Wavelength (nm) Wavelength (nm)

Figure 7. Absorption spectra in the Soret and Q-band regions of the molten-globule (MG) form and the forms unfolded by acid (UaCid) and urea (Uurea) of zinc cytochrome c monitored over time. Conditions: (a) 4.5 pM Zncyt (MG) at pH 2.0 and ja = 1.500 M over a period of

31 h; (b) 10.0 pM Zncyt (MG) at pH 2.0 and = 0.500 M over 12 h; (c) 4.5 pM Zncyt (Uacid) at pH 2.0 and |x = 20 mM over 18 h; and (d) 5.0 pM Zncyt (Uurea) at pH 7.00, *i = 20 mM, and 8.0 M urea over 9 h. Note the break in the wavelength axis and the different absorbance scales. 74

/// • F*. J "md / / / * I ° MG /// • Fhigh

* 150

100

T 0 20 40 60 80 100 120 20 40 60 80 100 120 140 160

3+ [Fe(CN)6 ] (gM) [Co(phen)3 l (gM)

20 30 40 SO 10 20 30 40 50 60 [Co(acac),] (jiM) tcyKIII)] (gM)

Figure 8. Dependence on quencher concentration of the quenching rate constant /fcq obtained using eq 8 for the reactions at 25 °C of the triplet state of zinc cytochrome c in the folded forms at ionic strengths of 20 mM (F|0W) and 1.500 M (Fhigh), molten-globule (MG) form, and forms unfolded by acid (UaCid) and by urea (Uurea) with the four quenchers shown. The solid lines are obtained from linear least-squares regression; their slopes yield the bimolecular rate constants £bim, given in Table 3. Invisible error bars are smaller than the symbol. The legend symbols are arranged from top to bottom in the same order as the slopes of the lines. Some plots overlap. See Figure 1 for experimental conditions. 75

Supporting Information

Zncyt - 0.12 Zncyt MG 1 F Hzcy hiflh H2cyt Flow - 0.10

- 0.08 8 c 8 0.8 - 0.06 i 1 < - 0.04

0.02

- 0.00 t 1 1 1 r 360 380 400 420 440 480 520 560 600 640 Wavelength (nm) Wavelength (nm)

Figure SI. Absorption spectra in the Soret and Q band regions of 5.0 zinc cytochrome c (Zncyt) in the MG (pH 2.0 and p. = 1.500 M) and Uacid (pH 2.0 and n = 20 mM) forms and the same two protein samples after several slow solvent exchanges by ultrafiltration into the folded Fiow (10 mM phosphate buffer at pH 7.00 and p. = 20 mM) and Fhigh (10 mM phosphate buffer at pH 7.00 and n = 1.500 M) forms. Absorption spectra of these protein samples after ultrafiltration is characteristic of metal-free porphyrin cytochrome c (H]cyt) confirming that demetalation of zinc cytochrome c occurs. 76

O E "O TE 0 1

x -10 Zncyt UKld o* Zncyt MG — .12 H2cyt Fhigh H2cyt Flow -14

190 200 210 220 230 240 250 260 Wavelength (nm) Figure S2. Far-UV circular dichroism spectra in the far-UV region of 5.0 |xM zinc cytochrome c (Zncyt) in the MG (pH 2.0 and p. = 1.500 M) and Uacjd (pH 2.0 and n = 20 mM) forms and the same two protein samples after several slow solvent exchanges by

ultrafiltration into the folded Fiow (10 mM phosphate buffer at pH 7.00 and p. = 20 mM) and Fhigh (10 mM phosphate buffer at pH 7.00 and p. = 1.500 M) forms. Circular dichroism spectra of these protein samples after ultrafiltration containing metal-free porphyrin cytochrome c (Hicyt) show recovery of native-like secondary structure upon conversion of forms UaCid into F|0W and MG into Fhjgh. 77

y 1.0

380 390 400 410 420 430 440 450 Wavelength (nm)

Figure S3. Conversion of 5.0 pM zinc cytochrome c from the Uacjd (pH 2.0 and g = 20 mM) to MG (pH 2.0 and p = 1.500 M) form effected by adding NaCl to the solution and monitored by absorbance changes in the Soret region. Clear isosbestic points persist at 398 nm and 417 nm as the ionic strength is raised from 20 to 110 mM. These isosbestic points become somewhat "smeared" upon going to 1.000 M. Spectra are corrected for dilution. 78

0.8

0) O 0.6 C

Io (O 0.4 <

0.2

0.0

360 380 400 420 440 460 Wavelength (nm)

Figure S4. Conversion of 4.0 |iM metal-free porphyrin cytochrome c (Hzcyt) into zinc cytochrome c (Zncyt) effected by adding 100 |*M zinc(H) acetate to the protein solution acidified by 10% acetic acid at 50°C and monitored by absorbance changes in the Soret region. Metalation of metal-free porphyrin cytochrome c forming zinc cytochrome c (Soret band at 423 nm) is nearly quantitative. 79

- 0.12

- 0.10

h 0.08 g 5 0.8 - - 0.06 o

- 0.04

- 0.00 360 380 400 420 440 480 520 560 600 640 Wavelength (nm) Wavelength (nm)

Figure S5. Absorption spectra in Soret and Q band regions of non-native UaCid (pH 2.0 and p, = 20 mM) and MG (pH 2.0 and p. = 1.500 M) forms of 10.0 pM iron(m) cytochrome c monitored over time. No change in absorbance is observed over a period of 2 and 5 days in the Uacid and MG forms, respectively. The stability of iron(IH) cytochrome c towards demetalation at pH 2 is evident and will not complicate analysis of the kinetics for reactions in which this protein is used as a quencher. 80

CHAPTER 4. GENERAL CONCLUSIONS

Because metalloproteins act as electron carriers in essential biological processes, such as respiration, photosynthesis, and metabolism, mechanisms of their electron-transfer reactions are being studied vigorously. The goal for many years has been to understand how protein structure and the structure of the redox center itself influence the rate constants for electron transfer between proteins. Toward this goal, we strive to understand how protein conformation modulates electron-transfer reactivity in heme proteins. We investigated photoinduced electron-transfer reactions of folded, partially unfolded, and completely unfolded zinc cytochrome c with various inorganic and protein electron acceptors.

In our studies of Zncyt in the folded conformation, photoinduced reactions of the excited 3Zncyt with iron(III) cytochrome c, iron(II) cytochrome c, metal-free porphyrin cytochrome c, and heme-free apocytochrome c revealed the mechanism of quenching. The replacement of heme iron by zinc(H) overcomes the difficulties in studying redox reactions between two heme proteins having similar absorption spectra. Quenching of the positively charged 3Zncyt by the positively charged reactants is promoted at high ionic strength, p. =

1500 mM, which shields electrostatic charge and weakens protein-protein repulsion.

Electron transfer from 3Zncyt to iron(III) cytochrome c is a more efficient quenching mechanism than energy transfer and enhanced radiationless decay at high ionic strength.

Oxidative quenching of 3Zncyt by iron(III) cytochrome c is proven by the observed formation of the electron-transfer intermediate Zncyt+. Iron-free porphyrin cytochrome c and also iron(H) cytochrome c quenches 3Zncyt by energy transfer, owing to the lack of any observable 81 transient radical-ions Zncyt* and Zncyt" and the good overlap of their absorption bands with the emission bands of 3Zncyt. Enhanced radiationless decay will not be an efficient mechanism of quenching 3Zncyt by any cytochrome c derivative used in this study.

To study the effect of protein conformation on reactivity, we compared the electron- transfer properties of 3Zncyt in the folded, molten-globule, and completely unfolded forms

3 3+ toward the following four oxidative quenchers: Fe(CN)ô ~, Co(acac)3, Co(phen)3 , and iron(m) cytochrome c. The observed bimolecular rate constants show electron-transfer reactivity depends mostly on electrostatic interactions and the degree of porphyrin exposure as the protein unfolds. Ionic strength and pH are chosen in some cases to bring out, but in most cases to minimize, effects of electrostatic interactions between Zncyt and the oxidative quenchers, so that effects of conformation on reactivity become discernible. The large increase in the observed bimolecular rate constants for the reactions of folded 3Zncyt and

3+ cationic quenchers Co(phen)3 and iron(HI) cytochrome c upon increasing ionic strength from 20 to 1500 mM shows the dominant role electrostatic repulsions play in controlling these reactions. Moreover, the even larger increase in the observed bimolecular rate constants for the reactions of 3Zncyt and cationic quenchers Co(phen)]3* and iron(m) cytochrome c at low ionic strength upon increasing the concentration of urea from 0 to 8 M shows the dominant role protein conformation plays in controlling these reactions. Using the electroneutral complex Co(acac)3 as a quencher, we eliminated the electrostatic effects and assessed only the consequences of porphyrin exposure upon partial and complete unfolding of the protein for the electron-transfer reactivity. The bimolecular rate constant for the reaction of 3Zncyt and Co(acac)] increases 10-fold upon partial unfolding into the molten- 82 globule form and approximately 50-fold upon complete unfolding of Zncyt. Electroneutral inorganic complexes are sensitive probes of the amount of partial and complete unfolding in zinc-substituted heme proteins. By measuring the chemical reactivity of proteins in various conformational forms, we can detect and characterize intermediates along the pathway of protein folding. A future application of our approach could be the detection of transient kinetic intermediates on the time scale of microseconds to seconds that arise during protein folding. 83

appendix. optimization of a bacterial

expression system for the soluble form of

turnip cytochrome F and its isolation

Introduction

Cytochrome/, designated cyt/, is an intrinsic membrane component of the

cytochrome ^complex that transfers electrons from the Rieske iron-sulfur protein to

plastocyanin and cytochrome c$. This 31-kDa protein is synthesized with a cleavable leader

sequence and is anchored in the thykaloid membrane by a single C-terminal transmembrane

helix. Cyt/contains the characteristic fingerprint sequence Cys-X-Y-Cys-His of c-type cytochromes, which is responsible for the covalent attachment of the heme group to the protein backbone through two thioether bonds. The crystal structure of the soluble form of cyt/from turnip chloroplasts has revealed three unprecedented features for c-type cytochromes (Martinez et al., 1994; Martinez et al., 1996). The overall structure of cyt/is elongated containing predominantly p-sheet character. Cyt/also contains two readily discernable domains, with the heme group covalently bound within the large domain. The third unprecedented aspect of cyt/is the heme ligation by the «-amino group of the amino- terminal residue Tyrl. This unusual mode of coordination may ensure a proper delay in the timing of protein refolding, preventing cyt/from properly folding until the signal peptide has been cut off by a peptidase located in the lumen. In addition, crystal structures of cyt/from green alga Chlamydomonas reinhardtii (Chi et al., 2000; Sainz et al., 2000) and 84 cyanobacterium Phormidium laminosum (Carrel 1 et. al, 1999) have been reported. Solution structures of the complex between cyt/and plastocyanin have also been determined using

NMR and rigid body molecular dynamics simulations (Ubbink et al., 1998; Crowley et al.,

2001).

Cyt/has been isolated from a variety of plant, algal, and cyanobacterial sources. For most higher plants (parsley, spinach, and tobacco) purification of cyt/results in the production of oligomeric or aggregated forms of the protein (Gray et al., 1994; Bendall,

1982). However, monomelic forms of cyt/ with no tendency to aggregate, have been isolated from the Cruciferae family, which includes mustard, radish, charlock, cabbage, and turnip. (Beoku-Betts & Sykes, 1985; Gray, 1978).

At the present time, commercially available cyt/is very expensive and large quantities needed for further NMR and kinetic experiments are difficult to obtain. Isolation of cyt /from higher plants is a tedious process with the risk of possible plant contaminants.

Therefore it would be desirable to produce higher plant cyt/in a bacterial expression system, capable of producing large quantities of protein in shorter time. Furthermore, production of cyt/mutants would be possible, leading to studies investigating electrostatic and hydrophobic interactions in protein-protein complexes.

At the time of this research (9/99 - 11/99), there were no published reports of expression systems for higher plant cyt/, although an expression system had already been developed for the cyanobacterial form of cyt/(Schlarb et al., 1999). Prior to my arrival at

Leiden University in Leiden, The Netherlands, development of a bacterial expression system for higher plant cyt /had just begun (Cornvik & Ubbink, 1999). The goal was to construct an 85

expression system for the soluble part of turnip (Brassica rapa) cyt/, residues 45-296, in

Escherichia coli. Since cyt/is a c-type cytochrome, heme lyase is required for covalent

attachment of heme to the protein backbone. In E. coli, heme lyase is located in the

periplasmic space, so it is essential to export cyt/there as well. This was achieved by

insertion of the nucleotide sequence of an efficient periplasmic signal peptide from azurin in front of the gene encoding cyt/. Moreover, isolation of cyt/is much easier after export into the periplasmic space (Ubbink & van Beeaman, 1992).

This report summarizes the optimization of soluble turnip cyt/expression in E. coli and its subsequent isolation and purification. Previous work in the laboratory at Leiden

University, Leiden, The Netherlands (Cornvik, T. & Ubbink, M.) showed plasmid pTCl expressed in E. coli strain JM109 under semi-anaerobic conditions at 30°C provided the highest expression levels of turnip cyt/. In continuation of this work, I investigated the effect of growth time, level of dissolved oxygen, and presence of pEC86 (a cassette of cytochrome c maturation genes of E. coli) on cyt/expression levels in a 30-L fermentor. Highest expression yields of cyt/are achieved after 60-72 hours under semi-anaerobic conditions.

Expression yields before isolation from growing cells are 0.9-1.0 mg/L. In addition, I optimized the recovery of cyt/after extraction from the periplasmic space by osmotic shock.

Cyt/yields after extraction by osmotic shock are typically 0.6-0.8 mg/L. Finally, I developed a procedure to isolate cyt/from periplasmic extracts using a combination of ion-exchange and gel filtration chromatography. The purification involves a DEAE Sepharose (batch mode), DEAE Sepharose (pH 6, cyt /reduced), CM Sephadex (pH 6, cyt / reduced), and

Superdex G75 (pH 6, cyt/reduced). The final yield from 30 L of culture was 10-12 mg cyt/ 86

with an absorbance ratio of A554/A277 of 0.95. During my stay in Leiden, nearly 50 mg of

pure cyt /was obtained. After this research was completed, an article describing an E. coli

expression system for soluble turnip cyt/and its isolation was published by another

laboratory (Gong et al., 2000).

Experimental

Expression. Plasmid pTCl (Figure 1), containing the gene encoding the soluble form of cyt/ was obtained from Dr. M. Ubbink (Comvik & Ubbink, 1999). Plasmid pEC86, a cassette of cytochrome c maturation genes of E. coli, was a gift from Dr. L. Thoeny-Meyer,

Microbiology Institute, ETH Zurich, Switzerland (Arslan et al, 1998). Competent cells of E. coli strain JM109 were transformed with plasmid pTCl and incubated overnight at 37°C in

1.5% Agar/LB plates supplemented with 100 pg/mL ampicillin. LB medium contained 10%

Difco tryptone peptone, 5% Difco yeast extract, and 10% sodium chloride. A single white colony was picked and diluted into 3 mL LB (in 10 mL sterile tube) supplemented with ampicillin and incubated 4-5 hours with shaking at 30°C. Fermentor medium (30 L) containing 300 g Difco tryptone peptone, 150 g Difco yeast extract, 240 g sodium chloride, and 3.0 g potassium nitrate was sterilized. The fermentor was inoculated with 250 mL 0.5 M fumarate (filter sterilized), 5.0 g ampicillin, and 3 mL JM109/pTCl preculture (slightly turbid). If available, 150 mL of a stock 0.2 g/50 mL Haemin solution was added

(McConville & Charles, 1979). Culture was grown at 30°C and pH 6.8 under semi-anaerobic conditions with dissolved O2 concentration regulated at 1.5%. When agitation rate reached

150 rpm (approximately 10 hours after inoculation), culture was induced with 1.5 g IPTG. 87

Figure 2 shows the fermentation conditions throughout the growth of the culture.

Periplasmic expression of cyt/during fermentation was determined via protein isolation by osmotic shock of the cells. A 10-mL sample from the fermentor was obtained (in

10-mL sterile tube) and centrifuged at 3300 rpm for 15 min. The pellet was resuspended in 1 mL sucrose buffer (30 mM Tris-HCl, 5 mM EDTA, 20% sucrose, pH 8) and incubated 20 minutes at room temperature. The cells were centrifuged at 3300 rpm for 15 min and absence of cyt /in supernatant (sucrose fraction) confirmed by absorbance spectroscopy. The pellet was resuspended in 1 mL cold Milli-Q water and incubated 20 min at 4°C. The cells were centrifuged at 3300 rpm for 15 min and presence of cyt/in supernatant (MilliQ fraction) confirmed by absorbance spectroscopy. Concentration of cyt/(mg/L) was obtained by using eq 1 where AA554 nm is the difference between the maximum A554 nm value and the value of

A554 nm obtained by extrapolating the baseline.

AAs54nm x2.8x10" = [cyt/] (in mg/L) (1) 31500 L 1

Cells were harvested from the fermentor and concentrated after approximately 60-72 hours of growth. It was determined that freezing the harvested cells and later thawing them significantly lowered the recovery of cyt/ Therefore, immediately after harvest, the cells were centrifuged at 6000 rpm in a JA-20 rotor for 25 min and the pellets resuspended in a total of 3 L sucrose buffer (a 5-L beaker was used). The cells were stirred at room temperature for 15 min after addition of 1 |iL DNase I. The cells were centrifuged at 6000 rpm for 25 min. The absence of cyt /in the supernatant (sucrose fraction) was confirmed by absorption spectroscopy. The pellets were resuspended in a total of 6 L cold Milli-Q water

(use two 5-L beakers) and stirred vigorously in a cold room at 4°C for 30 min. The cells 88

were centrifuged at 6000 rpm for 25 min and the supernatant (MilliQ fraction) was collected.

Figure 3 shows the absorption spectrum of cyt / present in the MilliQ fraction. Harvesting

the cells and osmotic shock required about 6 hours.

Purification. Initial extraction of cyt/after osmotic shock was accomplished by

adding 250 mL packed volume of Pharmacia DEAE Sepharose Fast Flow chromatography

resin (pre-equilibrated in Milli-Q water, pH 6.5) to 6 L of the MilliQ fraction and stirring a

few hours in a cold room. Cyt/was bound by the chromatography resin. Excess solution

was removed from the chromatography resin through a glass filter. Absence of cyt/in the

eluent was confirmed by absorption spectroscopy. A 350-mL slurry of the chromatography

resin was prepared and poured into large batch chromatography column from Pharmacia (2.6

cm internal diameter). Bound cyt/ was eluted using a solution of 10 mM MES buffer and

100 mM NaCl at pH 6.5. The flow rate was 3 mL/min. The best fractions of cyt/(as

determined by the purity ratio A554/A277 > 0.03) were pooled and 1 mM ascorbate was added

to ensure cyt /remained in the reduced iron(H) form. Cyt/in the oxidized form decomposes

readily. The solution of cyt/was exchanged into 10 mM MES buffer at pH 6.5 using an

Amicon cell. After concentration of the cyt/solution using an Amicon cell, 1 mM ascorbate

was added. The cyt/solution was frozen at -80°C until needed. Figure 4 shows the

absorption spectrum of pooled cyt/after this DEAE batch column.

A proper DEAE ion exchange column (internal diameter 1.6 cm), pre-equilibrated

with 10 mM MES buffer pH 6.5, was prepared. The cyt/solution recovered from the DEAE batch column was diluted with 10 mM MES buffer at pH 6.5 and 1 gL DNase I was added.

The cyt /solution was loaded onto the DEAE column at a flow rate of 3 mL/min. Cyt/was 89

eluted overnight with a linear salt gradient at a flow rate of 3 mL/min (Buffer A: 2.2 L

solution of 10 mM MES buffer at pH 6.5; Buffer B: 2.2 L solution of 10 mM MES buffer

and 60 mM NaCl at pH 6.5). The best fractions of cyt/(as determined by the purity ratio

A554/A277 > 0.2) were pooled and 1 mM ascorbate was added. The solution of cyt/was

exchanged into 10 mM MES buffer at pH 6.0 using an Amicon cell.

A proper Pharmacia CM Sephadex ion exchange column (1.6 cm internal diameter),

pre-equilibrated with 10 mM MES buffer at pH 6.0, was prepared. Cyt/ was eluted

overnight with a linear salt gradient at a flow rate of 3 mL/min (Buffer A: a 700 mL solution

of 10 mM MES buffer at pH 6.0; Buffer B: a 1 L solution of 10 mM MES buffer and 300

mM NaCl at pH 6.5). The best fractions of cyt/(as determined by the purity ratio A554/A277

> 0.4) were pooled and 1 mM ascorbate was added. The solution of cyt/was exchanged into

10 mM MES buffer at pH 6.0 using an Amicon cell. The CM Sephadex chromatography

resin binds cyt/very strongly, so short columns and higher salt gradients are recommended.

A Pharmacia Superdex G75 commercial FPLC gel filtration column, pre-equilibrated

with a solution of 10 mM MES buffer, 100 mM NaCl, and ImM ascorbate at pH 6.0 was

used. Samples (2-mL sample loop) of concentrated cyt/were injected onto the column and

eluted at a flow rate of 1 mL/min. The best fractions of cyt/(as determined by the purity

ratio A554/A277 ~ 0.9-1.0) were pooled and 1 mM ascorbate was added. The cyt /solution

was concentrated in Amicon cell.

Additional purification attempts used Pharmacia SP Sepharose and HiTrap Q

Sepharose commercial FPLC ion exchange columns. After equilibrating a SP Sepharose column with 20 mM MES buffer at pH 6.5, cyt/was loaded. However, cyt/bound poorly to 90 the chromatography resin and eluted soon after increasing the flow rate to 3 mL/min. After equilibrating a Q Sepharose column with 10 mM MES buffer at pH 6.5, cyt/was loaded. A linear gradient of 0 to 60 mM NaCl was used to elute cyt/ At a flow rate of 5 mL/min, the cyt/band smeared, so flow rate was adjusted to 2 mL/min. Again, cyt/bound poorly to the column and quickly eluted at 24% 60 mM NaCl.

Results and Discussion

Expression. The expression yields of turnip cyt/in E. coli as the culture is growing in the fermentor are shown in the Figure 5. Remarkably, maximum cyt/expression yields plateau at approximately 50 to 55 hours. This suggests that in this expression system, the holoenzyme is produced during the stationary phase.

Tables 1 and 2 summarize cyt/expression and osmotic shock conditions. Both tables illustrate the difficulties in maximizing expression yields and isolating cyt/from cells by osmotic shock. In Table 1, the correlation of longer growth time to higher expression yield of cyt/in the growing culture is clear. However, growth conditions for this bacterial expression system may still be improved. Surprisingly, semi-anaerobic and aerobic growth conditions both show similar yields (-15 mg cyt f) at shorter growth times (30 hours).

However, it is not clear whether this will be true at longer growth times. Moreover, incorporation of plasmid pEC86 could not improve yields of cyt /in this bacterial expression system. Actually, pEC86 increased formation of an unknown contaminating heme protein with Soret absorbance maximum at 408 nm (Figure 6).

The problem of low percent recovery of cyt/in the MilliQ fraction after osmotic shock was troubling. It was observed that when cells are frozen after harvest, lower yields of cyt/are recovered in the MilliQ fraction (-30% recovery). Cells may be disrupted upon freezing and when osmotic shock is subsequently performed, cyt/ was observed in both the sucrose and MilliQ fractions. In cases where cells were not frozen after harvest, higher yields of cyt/were recovered in the MilliQ fraction (80-100% recovery), and no cyt/was observed in the sucrose fraction. Additionally, larger volumes of Milli-Q water (6 L per 30-L fermentor instead of 3 L) helped to improve cyt/recovery (see Table 2).

Purification. Initial extraction of cyt/from the MilliQ fraction utilized CM or

DEAE batch ion exchange columns. DEAE performs better than CM in binding cyt/in the

MilliQ fraction and in reproducibility. Tables 3 and 4 summarize the percent recovery and purity ratio for each fermentation. See Figure 4 for an absorption spectrum of cyt/after the

DEAE batch column.

The next step in purification utilized DEAE ion exchange chromatography. The first six and last four fermentations were pooled and purified using two different DEAE columns.

Table 5 summarizes the results.

Further purification of cyt/was done in two ways: A) Cyt/recovered off DEAE column #1 (28 mg) was purified first using CM ion exchange chromatography and then gel filtration chromatography on the FPLC. B) Cyt/recovered off DEAE column #2 (65 mg) was purified first by gel filtration chromatography on the FPLC and then by CM ion exchange chromatography.

Tables 6 and 7 summarize pathway A. A major problem with CM column #1 was that cyt/bound strongly to column and could not be easily eluted. Percent recovery was low 92

since cyt/oxidized and denatured on the column. CM column #2 utilized a shorter column

and higher salt gradient to overcome these difficulties (Table 6).

The final step in pathway A is gel filtration chromatography. The best fractions of cyt

/ (as determined by the purity ratio) were pooled and concentrated, loaded onto the gel

filtration column, and eluted. Percent recovery is low owing to significant loss of protein

upon injection into the sample loop. However, the purity ratio is excellent for future NMR

and kinetic measurements (Table 7).

Tables 8 and 9 summarize pathway B. Three samples of cyt / with different purity

ratios were obtained from DEAE column #2: "best band", "tails best band", and "poor

band". These samples of cyt/were purified separately by gel filtration chromatography. In

chromatography runs 2 through 5,1 mM ascorbate was added to the buffer. Owing to the

strong absorbance of ascorbate at 280 nm, purity ratios could not be determined after

purification by gel filtration. See Figure 7 for absorption spectrum of cyt/after gel filtration chromatography.

The final step in purification of cyt/following pathway B was CM ion exchange chromatography. Samples of cyt/with the highest purity ratios were pooled and concentrated, loaded onto the CM ion exchange column, and eluted. We obtained 47 mg of cyt/with an excellent purity ratio for future NMR and kinetic experiments. See the Figure 8 for an absorption spectrum of pure cyt /

Throughout the entire purification procedure, an unknown contaminating heme protein with a Soret band at 408 nm was present. Cation (CM and SP) and anion exchange

(DEAE and Q) chromatography could not sufficiently separate cyt /from this unknown 93

protein. In addition, gel filtration could not sufficiently separate these two proteins. The

contaminating heme protein eluted right after cyt/. Based on the above results, the molecular

weight, charge, and binding properties of these two proteins are very similar. Unfortunately,

loss of cyt/during purification in fractions containing the contaminating protein is inevitable.

Further characterization of this unknown heme protein should provide clues for more

efficient separation from cyt/.

Conclusions

A bacterial expression system for the soluble form of turnip cyt/has been achieved.

Expression yields before isolation from growing cells are 0.9-1.0 mg/L. However, cyt/

yields after extraction from the periplasmic space by osmotic shock are typically 0.6-0.8

mg/L. Cyt /is purified from periplasmic extracts by a combination of ion exchange and gel

filtration chromatography. The purification sequence is DEAE Sepharose (batch mode),

DEAE Sepharose (pH 6, cyt/reduced), CM Sephadex (pH 6, cyt/reduced), and Superdex

G75 (pH 6, cyt/reduced). The final yield from a 30-L culture is 10-12 mg cyt/with an absorbance ratio A554/A277 of 0.95.

Acknowledgements. This work was supported by and completed under the guidance of Dr. Marcellus Ubbink and Professor Gerard Canters at the Leiden Institute of Chemistry,

Leiden University, Leiden, The Netherlands. Plasmid pTCl was kindly provided by Tobias

Cornvik and Dr. Marcellus Ubbink. Plasmid pEC86 was a gift from Professor Linda

Thoeny-Meyer, Microbiology Institute, ETH Zurich, Switzerland. 94

References

Arslan, E.; Shulz, H.; Zufferey, R.; Kuenzler, P.; Thoeny-Meyer, L. "Overproduction of the

Bradyrhizobium japonicum c-type cytochromes subunits of the cbb3 oxidase in

Escherichia coli." Biophys. Res. Commun. 1998, 251, 744.

Bendall, D. S. Biochim. Biophys. Acta 1982, 683, 119.

Beoku-Betts D.; Sykes G. A. "Kinetic Studies on Electron-Transfer Reactions of Brassica

oleracea Cytochrome/with Inorganic Oxidants of Varying Charge (-5 to +3)." Inorg.

Chem. 1985, 24, 1142.

Carrell, C. J.; Schlarb, B. G.; Bendall, D. S.; Howe, C. J.; Cramer, W. A.; Smith, J. L.

"Structure of the Soluble Domain of Cytochrome / from the Cyanobacterium

Phormidium Laminosum." Biochemistry 1999, 38, 9590.

Chi, Y.-I.; Huang, L.S.; Zhang, Z.; Fernandez-Velasco, J. G.; Berry, E. A. "X-ray Structure

of a Truncated Form of Cytochrome/from Chlamydomonas Reinhardtii."

Biochemistry 2000, 39, 7689.

Comvik, T.; Ubbink. M. "Development of an Expression System for Turnip Cytochrome/in

Escherichia coli." unpublished research report, Leiden Institute of Chemistry, Leiden,

The Netherlands, 1999.

Crowley, P. B.; Otting G.; Schlarb-Ridley B. G.; Canters G. W.; Ubbink M. "Hydrophobic

Interactions in a Cyanobacterial Plastocyanin-Cytochrome/Complex." J. Am. Chem.

Soc., 2001, 123, 10444.

Gong, X.-S.; Wen, J. Q.; Gray, J. C. "The Role of Amino-Acid Residues in the Hydrophobic

Patch Surrounding the Haem Group of Cytochrome/in the Interaction with Plastocyanin." Eur. J. Biochem., 267, 1732.

Gray, J. C. "Purification and Properties of Cytochrome/from Charlock." Eur. J. Biochem.

1978, 82, 133.

Gray, J. C.; Rochford, R. J.; Packman, L. C. "Proteolytic Removal of the C-Terminal

Transmembrane Region of Cytochrome/During Extraction from Turnip and

Charlock Leaves Generates a Water-Soluble Monomeric Form of the Protein." Eur. J.

Biochem. 1994, 223,481.

Martinez, S. E.; Huang, D.; Ponomarev, M.; Cramer, W. A.; Smith, J. L. "The Heme Redox

Center of Chloroplast Cytochrome/is Linked to a Buried Five-Water Chain." Protein

Sci. 1996, 5, 1081.

Martinez, S. E.; Huang, D.; Szczepaniak, A.; Cramer, W. A.; Smith, J. L. "Crystal Structure

of Chloroplast Cytochrome / Reveals a Novel Cytochrome Fold and Unexpected

Heme Ligation." Structure 1994, 2,95.

McConville, M. L.; Charles, H. P. "Mutants of Escherichia coli K12 permeable to heamin."

J. Gen. Microbiol. 1979, 113, 165.

Navarro J. A.; Hervas M.; De la Rosa M. A.; "Co-Evoloution of Cytochrome c<$ and

Plastocyanin, Mobile Proteins Transferring Electrons from Cytochrome bff to

Photosystem I." JBIC1997,2, 11.

Sainz, G.; Carrell, C. J.; Ponamarev, M. V.; Soriano, G. M.; Cramer, W. A.; Smith, J. L.

"Interruption of the Internal Water Chain of Cytochrome/Impairs Photosynthetic

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Schlarb B. G.; Wagner M.J.; Vijgenboom E.; Ubbink M.; Bendall D. S.; Howe C.J. 96

"Expression of Plastocyanin and Cytochrome/of the Cyanobacterium Phormidium

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Tables and Figures

Table 1. Fermentor yields and percent recovery of cyt/in MilliQ fractions

Growth Yield Cyt / Yield Cyt/in Date Plasmid & % Recovery Time in Fermentor MilliQ Fraction Harvested Conditions of Cyt / (hours) (mg) (mg)

pTCl+pEC86 10/02/99 34 15.5 4.7 30.4 semi-anaerobic

pTCl+pEC86 10/06/99 33 19.2 6.2 32.3 semi-anaerobic

pTCl+pEC86 10/08/99 31 15.3 3.2 20.9 aerobic

pTCl+pEC86 10/12/99 30 13.2 3.6 27.1 aerobic

pTCl 10/15/99 32 12.4 6.1 72.0 semi-anaerobic

pTCl 10/22/99 45 20.0 16.4 81.9 semi-anaerobic

pTCl 10/25/99 71 20.2 20.1 99.4 semi-anaerobic

pTCl 10/29/99 68 29.3 24.6 84.0 semi-anaerobic

pTCl 11/02/99 74 29.0 26.5 91.4 semi-anaerobic

pTCl 11/05/99 71 23.7 25.9 109.2 semi-anaerobic

pTCl 11/08/99 73 21.8 10.0 45.9 semi-anaerobic 98

Table 2. Osmotic shock conditions and yields of cyt/in MilliQ Fractions

Date MilliQ Used Yield Cyt/ Plasmid Comments Harvested (L) (mg)

10/02/99 pTCl+pEC86 1 1.3 1/3 pellet frozen -80°C

1 1.3 1/3 pellet frozen -80°C

1 2.2 1/3 pellet frozen -80°C

10/06/99 pTCl+pEC86 1 2.4 1/3 pellet frozen -80°C

1 2.0 1/3 pellet frozen -80°C

1 1.9 1/3 pellet frozen -80°C

10/08/99 pTCl+pEC86 1 1.2 1/3 pellet frozen -80°C

2 2.0 2/3 pellet frozen -80°C

10/12/99 pTCl+pEC86 2 3.1 2/3 pellet not frozen

1 0.5 1/3 pellet frozen -80°C

10/15/99 pTCl 2 2.4 1/3 pellet frozen -80°C

2 1.9 1/3 pellet frozen -80°C

2 1.8 1/3 pellet frozen -80°C

10/22/99 pTCl 4 11.1 2/3 pellet not frozen

2 5.3 1/3 pellet frozen -20°C

10/25/99 pTCl 6 20.1 3/3 pellet not frozen

10/29/99 pTCl 6 24.6 3/3 pellet not frozen

11/01/99 pTCl 6 26.5 3/3 pellet not frozen

11/05/99 pTCl 6 25.9 3/3 pellet not frozen

11/08/99 pTCl 6 10.0 3/3 pellet not frozen 99

Table 3. Percent recovery of cyt/from MilliQ fraction after an initial DEAE batch column

(first six fermentation results combined)

Cyt/eluted Dale Plasmid/ Cyt/added to Purity Ratio % Recovery from columns Harvested Conditions columns (mg) (mg) A55VA277 of Cyt/

Six Earlier pTCl + pEC86 40.2 31.5 78.4 Fermentors

Table 4. Percent recovery of cyt/from MilliQ fraction after an initial DEAE batch column

(last 4 fermentations separately)

Cyt/eluted Date Plasmid/ Cyt/added to Purity Ratio % Recovery from column Harvested Conditions column (mg) (mg) A554/A277 of Cyt /

PTCl 10/25 20.1 18.0 89.6 semi-anaerobic PTCl 10/29 24.6 19.6 79.7 semi-anaerobic PTCl 11/02 26.5 22.7 0.03 85.7 semi-anaerobic PTCl 11/05 25.9 20.7 0.02 79.9 semi-anaerobic 100

Table 5. Percent recovery of cyt/after a proper DEAE column. The first six and last four fermentations were pooled and loaded onto separate columns.

DEAE Cyt/ Cyt/added to Cyt/eluted from Purity Ratio % Recovery of column Fermentations column (mg) column (mg) AssVAm Cyt/

First 6 31.5 27.8 0.2 88.3

64 8 Last4 81 . . " . 0.1-0.3 80.0 best band

4.6 0.05 - 0.08 tails best band

8.4 0.02-0.03 poor band

Table 6. Percent recovery of cyt/from pathway A after a CM column.

CM Cyt / Cyt/added to Cyt/eluted from Purity Ratio % Recovery

column Fermentations column (mg) column (mg) A 554/A 277 of Cyt /

1 First 6 27.8 11.7 0.15 42.1

9.0 2 First 6 11.7 0.4 76.9 best band 101

Table 7. Percent recovery of cyt/from pathway A after a final gel filtration column.

Gel Cyt/added to Cyt/eluted from Purity Ratio % Recovery Cyt / Filtration column (mg) column (mg) A55VA277 of Cyt/ 1 0 SO 1 2 mL best band 9.0 1.8 © 20.0

Table 8. Percent recovery of cyt/from pathway B after a gel filtration column.

Gel Cyt/added to Cyt/eluted from Purity Ratio '% Recovery Cyt / Filtration column (mg) column (mg) A554/A277 of Cyt /

1 2 mL best band 19.9 15.3 0.3 - 0.4 76.9

2 2 mL best band 26.9 21.9 Unk 81.4

1 mL best band + 3 1 mL tails of best 15.6 11.8 Unk 75.6 band

4 2 mL tails best band 11.8 8.6 Unk 72.9

5 2 mL poor band 8.4 0.0 Unk 0.0 102

Table 9. Percent recovery of cyt f from pathway B after a final CM column.

rM , r t f Cyt/added to Cyt/eluted from Purity Ratio % Recovery column vyi/ column (mg) column (mg) AssJAzn of Cyt/

L ^°m.geI 57.6 47.0 0.87 - 0.95 81.5 filtration

2.4 0.4 103

T7 Promotor EcoRI

lacZ signal

cytf

pTC1 3667 bp

lacZ amp

f1 ori

Figure 1. Plasmid pTC 1 containing the gene encoding the truncated soluble form of cyt f 104

Harvest Pallet 110199 - pTC1 cyt f Sti.*"*-*'- /tv\A.tVT»b• C 404)0 100.0 8.00

M.00 7.20

3(0.0 32.00 60.0 6.40

240.0 40.0 5.60

24.00 20.0 4.80

20.00 4.00 00:00:00 11:48:00 37:30:00 f

Figure 2. Semi-anaerobic fermentation conditions (pH, temperature, agitation rate, and dissolved oxygen) versus growth time for expression of cyt/in E. coli. 105

0.06 -

0.05 -

0.04 - i 1 0.03 - <

0.02 -

0.01 -

0.00

400 450 500 550 600 Wavelength (nm)

Figure 3. Absorption spectrum of cyt/in MilliQ fraction after osmotic shock. 106

400 450 500 550 600 Wavelength (nm)

Figure 4. Absorption spectrum of cyt/after DEAE batch column. 107

30 -

25 -

E 20 -

10 -

0 10 20 30 40 50 60 70 80

Growth Time (hours)

Figure 5. Expression yields of cyt/in culture as growth time increases. 108

Due: 27/10W Time: 03:18:2»

pTC( + ^ Ae-vot'C.

MMi: 5, pZA-cA'dirs,^

0.14 j

0 12 _

0.11

0.10 J A

009 I

0.08 I

0.07 I

0.06 j

0.046

4000 420 440 460 480 500 520 540 560 580 600.0 NM

I0I2MOA.SP - 13/10/99 - MilliQ Fraction 2/3 Pellet 101299 not frozen

Figure 6. Absorption spectrum of MilliQ fraction after osmotic shock. Aerobic expression of plasmids pTCl and pEC86 showing increased presence of unknown contaminating heme protein with Soret band at 408 nm. 109

0.4

0.3 -

0.2 -

?.o <

0.0 -

300 400 500 600 Wavelength (nm)

Figure 7. Absorption spectrum of cyt/after gel filtration and before final CM column. 110

0.30 -

0.25 -

0.20 - 1 SU 1 0.15 - <

0.10 -

0.05 -

0.00 -

300 400 500 600 Wavelength (nm)

Figure 8. Absorption spectrum of pure turnip cyt/. Ill

acknowledgments

I am deeply indebted to Professor Nenad M. Kostic for his guidance, persistence, and

encouragement throughout my graduate career. He has provided many wonderful scientific,

professional, and personal opportunities to develop into the scholar I always wanted to be. I

wish to thank the past and present members of the Kostic group for their knowledge, advice,

and friendship. I greatly appreciate the hospitality and guidance of Dr. Marcellus Ubbink,

Professor Gerard Canters, and Professor Sabeeha Merchant during my visits to Leiden

University, The Netherlands and UCLA. I would like to thank the members of my POS committee and the faculty members of the Inorganic Division of the Department of

Chemistry at Iowa State University for their teaching, encouragement, and advice. I acknowledge the Department of Chemistry at Iowa State University for support through a

GAANN Fellowship and teaching and research assistantships. I would like to thank my parents for their encouragement to pursue my dreams.

To my wife Rachel, I will always remember the friendship, encouragement, patience, sacrifice, and unconditional love you have provided me these last ten years. I owe you everything.